Interferon-induced human protein in pure form, monoclonal antibodies thereto and test kits containing these antibodies

ABSTRACT

The invention relates to purified proteins induced in human cells by interferon α or β, RNAs, DNAs and hybrid vectors coding for said proteins, hosts transformed with such a hybrid vector, processes for the preparation and purification of these proteins, DNAs, vectors and hosts, monoclonal antibodies specific to these proteins, monoclonal antibody derivatives, hybridoma cell lines secreting these monoclonal antibodies, the use of the monoclonal antibodies and their derivatives in the qualitative and quantitative determination of these proteins, test kits containing the monoclonal antibodies, and pharmaceutical preparations containing said proteins. A protein of the invention shows antiviral properties ascribed to interferons and may be a valuable indicator of the cell response to an interferon therapy.

This application is a divisional application of pending U.S. applicationSer. No. 08/941,928 filed Oct. 1, 1997, now U.S. Pat. No. 5,869,264which is a divisional of Ser. No. 08/444,344 filed May 18, 1995, nowU.S. Pat. No. 5,739,290, which is a divisional of Ser. No. 08/1258,902,filed Jun. 13, 1994, now U.S. Pat. No. 5,466,585 which is a Continuationof Ser. No. 07/983,177, filed Nov. 30, 1992, now abandoned which is adivisional of Ser. No. 07/810,580, filed Dec. 19, 1991 now U.S. Pat. No.5,198,350 which is a Continuation of Ser. No. 07/497,748, filed Mar. 19,1990, now abandoned, which is a Continuation of Ser. No. 07/037,754,filed Apr. 13, 1987, now abandoned.

FIELD OF THE INVENTION

The invention relates to purified proteins induced in human cells byinterferon α or β, RNAs, DNAs and hybrid vectors coding for saidproteins, hosts transformed with such a hybrid vector, processes for thepreparation and purification of these proteins, DNAs, vectors and hosts,monoclonal antibodies specific to these proteins, monoclonal antibodyderivatives, hybridoma cell lines secreting these monoclonal antibodies,the use of the monoclonal antibodies and their derivatives in thequalitative and quantitative determination of these proteins, test kitscontaining the monoclonal antibodies, and pharmaceutical preparationscontaining said proteins.

BACKGROUND OF THE INVENTION

Interferons are a class of naturally occurring proteins which showpromise in the defense against viral infections and in tumor growthcontrol. They seem to interfere with cellular functions necessary forviral replication and to inhibit cellular growth by pleiotropic effectswhich are not yet understood at the molecular level. Furthermore,interferons stimulate the activity of natural killer (NK) cells, andwithin a complicated network of lymphokine interactions, modulate theactivity of macrophages, B- and T-cells.

A set of induced proteins may be involved in the antiviral,antiproliferative and immunomodulatory activities of interferons. Inmammalian cells interferons induce the synthesis of several proteinsthat are not detected or do exist at much lower concentrations inuntreated cells. Some of these induced proteins have been widely studied[review: P. Lengyel, Annu. Rev. Biochem. 51, 251 (1982)], but are stillpoorly characterized. Interferon-treated cells of both human and mouseorigin contain elevated levels of 2′,5′-oligoiso-adenylate synthetaseand protein kinase activities. The synthesis and properties of the mouseprotein Mx induced by interferon α and β have been studied in detail [P.Staeheli et al., Cell 44, 147 (1986)]. This protein is associated with ahighly efficient and specific antiviral resistance to influenza viruses[M. A. Horisberger et al., Proc. Natl. Acad. Sci. USA 80, 1910 (1983);M. A. Horisberger & H. K. Hochkeppel, J. Biol. Chem. 260, 1730 (1985)].A related human protein was detected in interferon-induced humanperipheral blood lymphocytes and fibroblasts [P. Staeheli & O. Haller,Mol. Cell. Biol. 5, 2150 (1985)]. A molecular weight of 80 kDa(kilo-Dalton) was found, and the protein was predominantly localized inthe cell cytoplasm, but otherwise the protein was not furthercharacterized or isolated.

The fast progress in recombinant DNA technology in recent years providesthe general methods for the preparation of proteins in large amountsindependent of the primary natural sources of such compounds.Identification of an mRNA or a DNA coding for the desired polypeptide iscrucial for the success of this approach. If (partial) amino acidsequence information is available, a chemically synthetized nucleic acidprobe may lead to the isolation of coding mRNA or DNA from a mixture ofmRNA derived from cells producing the desired polypeptides or from a DNAlibrary, respectively. Although many examples for the isolation of anmRNA or DNA coding for a desired polypeptide have so far become knownand the general procedure has been described in principle, each newspecific problem requires adaption of the technique to the particularcase.

Once a complementary or genomic DNA coding for the desired polypeptideis at hand, preparation of suitable expression vectors, transformationof hosts with these vectors, fermentation of transformed hosts andisolation of the expressed polypeptide follows standard procedures. Hereagain, these procedures must be adapted to the particular problem inorder to get stable incorporation of the DNA and sufficiently highexpression of the desired polypeptide in a chosen host organism, andacceptable yields of pure, biologically active isolated protein.

Furthermore recombinant DNA technology allows one to produce polypeptidevariants by mutating or otherwise altering the coding DNA incorporatedin a host organism, thereby enlarging the potential applications of anactive principle found in a single polypeptide structure in nature.

Proteins induced by interferon are important in diagnosis, diseasemanagement and therapy in two respects: On one hand they may exert someof the beneficial properties such as antiviral or antiproliferativeactivities ascribed to interferons, but without the unwanted sideeffects of interferons, on the other hand they may be valuableindicators of the cell response to an interferon therapy. Antibodies tosuch interferon-induced proteins allow the qualitative and quantitativedetermination of these proteins and therefore are indispensable means inthe surveillance of a therapy with these proteins or with interferons.

Polyclonal and monoclonal antibodies to the mouse Mx protein induced byinterferons are known [P. Staeheli & O. Haller, Mol. Cell. Biol. 5, 2150(1985)]. One of these shows also a weak cross-reactivity to a humaninterferon-induced protein, however, non-specific cross-reaction cannotbe excluded.

OBJECT OF THE INVENTION

It is an object of the present invention to provide pure proteinsrelated to or identical with those found in human cells induced byinterferon α or β. The problem of industrial synthesis of such proteinscan be solved by the methods of recombinant DNA technology. A furtherobject of the present invention is therefore to provide mRNAs, DNAs andhybrid vectors coding for said proteins, and hosts transformed with sucha vector.

Further objects are processes for the preparation and purification ofsaid proteins, mRNAs, DNAs and hybrid vectors, and processes for tiepreparation of hosts containing said hybrid vectors.

It is another object of the invention to provide monoclonal antibodiesas diagnostic means for the qualitative and quantitative determinationof said proteins, hybridomas secreting such antibodies, and processesfor the preparation of these antibodies and hybridomas, furthermorepharmaceutical preparations containing said proteins.

DESCRIPTION OF THE INVENTION

The invention relates to essentially pure proteins, characterized by

(1) their presence in human cells induced by interferon α or β, but notin untreated cells to a reasonable extent,

(2) a molecular weight of approximately 78 kDa as determined by sodiumdodecyl sulfate polyacrylamide gel electrophotesis (SDS-PAGE),

(3) an isoelectric point of approximately 6.3,

(4) a partial N-terminal amino acid sequence

(SEQ ID NO.1)                  5                   10Val-Val-Ser-Glu-Val-Asp-Ile-Ala-Lys-Ala-.

In particular, these proteins are found in Namalwa or human embryonicforeskin cells treated with a recombinant interferon α/B, α/D, α/F orinterferon α/B-D hybrids.

Particularly preferred is a protein with a partial N-terminal amino acidsequence

                 5                   10                15 (SEQ ID NO.2)Val-Val-Ser-Glu-Val-Asp-Ile-Ala-Lys-Ala-Asp-Pro-Ala-Ala-Ala-Ser-             20                  25                  30His-Pro-Leu-Leu-Leu-Asn-Gly-Asp-Ala-Thr-Val-Ala-Gln-Lys-Asn-Pro-         35                  40                  45Gly-Ser-Val-Ala-Glu-Asn-Asn-Leu-Cys-Ser-Gln-Tyr-Glu-Glu-Lys-Val-     50                  55 Arg-Pro-Gys-Ile-Asp-Leu-Ile-Asp-.

The molecular weight of 78 kDa is based on SDS-PAGE with the usualmolecular weight markers. However, it can be inferred from work with therelated mouse protein Mx [P. Staeheli et al., Cell 44, 147 (1986))] thatthe actual molecular weight is lower, probably around 72 kDa.

The proteins are further characterized by the amino acid composition asdetermined by a total amino acid analysis based on a molecular weight of72 kDa (Table 1). The range of actual number of amino acids of theanalyzed proteins given in Table 1 is calculated from the uncertainty(standard deviation) of the method of analysis.

TABLE 1 Amino acid composition amount actual number of amino aciddetermined amino acids (range) Asx (Aspartic acid/Asparagine) 56.8 54-60Glx (Glutamic acid/Glutamine) 96.1  91-101 Ser (Serine) 39.3 37-41 Thr(Threonine) 32.2 30-34 Gly (Glycine) 43.4 41-46 Ala (Alanine) 47.2 45-50Arg (Arginine) 38.2 36-40 Pro (Proline) 26.0 24-28 Val (Valine) 39.938-42 Met (Methionine) 18.0 17-19 Ile (Isoleucine) 43.1 41-46 Leu(Leucine) 68.1 64-72 Trp (Tryptophan) 0 0-3 Phe (Phenylalanine) 25.424-27 Cys (Cysteine) 5.9 5-7 Lys (Lysine) 47.5 45-50 His (Histidine)12.9 12-14 Tyr (Tyrosine) 11.8 11-13

The invention relates also to a process for the preparation of such aprotein, characterized in that cells producing said protein are culturedand the protein is isolated from the cell supernatant or cell lysismixture and purified by precipitation and chromatographic methods.

In particular, the invention relates to a process for the preparation ofsuch a protein in purified form, characterized in that human cells,preferably cells of a continous human cell line, e.g. Namalwa cells orembryonic foreskin cells, are cultured in the presence of humaninterferon α or β, e.g. natural human interferon α or recombinant humaninterferon α, such as interferon α/B, α/D, α/F or α/B-D hybrids, thecells are lysed, the proteins in the supernatants are precipitated, e.g.by addition of ammonium sulfate, then separated by preparative gelelectrophoresis, and the protein of an apparent molecular weight ofapproximately 78 kDa is eluted, e.g. by electrodialysis.

Human cells useful for the preparation of the proteins of the inventionare normal lymphocytes, macrophages or monocytes, lymphoblastoid cells,e.g. Namalwa cells, or human embryonic foreskin cells from a diploidcell line.

Preferably, Namalwa cells are cultured in the usual cell growth media,e.g. RPMI 1640 medium supplemented with vitamins and/or hormones, forexample in the form of fetal calf serum, and optionally antibiotics. Atthe end of exponential growth, the cells are incubated with recombinantinterferon a, e.g. α/B subtype or α/B-D hybrids, in concentrationsranging from 5×10⁵ to 10⁷ cells per ml and 2000 to 10,000 internationalinterferon units per ml, preferably around 37° C.

Any of the known natural or recombinant interferon α or β may be used toinduce the production of the proteins of the invention, e.g. therecombinant interferons described in the patent applications EP 28 033,EP 32 134, EP 43 908, EP 72 541 or EP 76 489.

The cells are harvested and lysed by the usual methods, e.g. by highsalt concentrations in buffered solution, and the protein precipitated,e.g. by addition of ammonium sulfate. The desired protein is ratherinsoluble in the usual physiological solvent systems.

The desired protein is purified by gel electrophoresis. Preferably thepreparative gel electrophoresis is performed twice, e.g. first to effectcrude separation from proteins with molecular weight less than 70 kDaand more than 85 kDa, then by two-dimensional separation of theresulting protein mixture, combining non-equilibrium pH gradientelectrophoresis with SDS-polyacrylamide gel electrophoresis. Inparticular, the protein mixture is applied to the acidic end of thenon-equilibrium pH gradient electrophoresis gel, which contains 2%ampholytes, pH 3-10, separated electrophoretically, then separated inthe second dimension on a slab gel containing 10-15%, preferably 12%,acrylamide and up to 0.5%, e.g. around 0.3%, bis-acrylamide.

Preferably, a protein of the invention is prepared by recombinant DNAtechnique comprising, for example, culturing a transformed hostexpressing the protein as defined hereinbefore under conditions whichallow expression of the heterologous polypeptide and isolating thedesired compound. More specifically, the desired protein is prepared by

a) isolating a DNA coding for the protein from a cDNA or a genomic DNAlibrary of human cells,

b) incorporating the DNA into an appropriate expression vector,

c) transferring the obtained hybrid vector into a recipient host,

d) selecting the transformed host from untransformed hosts, e.g. byculturing under conditions under which only the transformed hostsurvives,

e) culturing the transformed host under conditions which allowexpression of the heterologous polypeptide, and

f) isolating the desired protein.

The steps involved in the preparation of these peptides by recombinantDNA technique will be discussed in more detail hereinbelow.

The invention relates also to DNAs coding for proteins as describedhereinbefore. In particular the invention concerns a DNA of the formula

       1                    10 (I), (SEQ ID NO.3 and 4)   MetValValSerGluValAspIleAlaLysAlaAspProAlaAlaAlaSerHisProLeu Z¹-ATGGTTGTTTCCGAAGTGGACATCGCAAAAGCTGATCCAGCTGCTGCATCCCACCCTCTA   1       10        20        30        40        50        60    20                            30   LeuLeuAsnGlyAspAlaThrValAlaGlnLysAsnProGlySerValAlaGluAsnAsn   TTACTGAATGGAGATGCTACTGTGGCCCAGAAAAATCCAGGCTCGGTGGCTGAGAACAAC           70        80        90       100       110       120    40                            50   LeuCysSerGlnTyrGluGluLysValArgProCysIleAspLeuIleAsp   CTGTGCAGCCAGTATGAGGAGAAGGTGCGCCCCTGCATCGACCTCATTGAC-Z²          130       140       150       160       170

wherein Z¹ is a 5′-end DNA residue of 12 nucleotides or more containinga promoter sequence, Z² is a DNA residue of 1700 or more codingnucleotides, a stop codon and optionally non-coding nucleotides at the3′-end, and Z¹ and Z² are optionally linked,

a DNA of formula I wherein one or more triplet codons are replaced byother triplet codons for the same amino acids, a double-stranded DNAconsisting of a DNA of formula I and of a complementary DNA thereto, andthat complementary DNA itself.

An example of a DNA of the formula I is e.g. the cDNA which is derivedfrom the mRNA of a human embryonic foreskin cell, of the formula

Z³-AGGTCTGTGATACCATTTAACTTGTTGACATTACTTTTATTTGAAGGAACGTATATTA (II), (SEQID NO.5 and 6)   -80        -70       -60       -50       -40       -30                               1                          10                           MetValValSerGluValAspIleAlaLysAlaAsp   GAGCTTACTTTGCAAAGAAGGAAGATGGTTGTTTCCGAAGTGGACATCGCAAAAGCTGAT     -20       -10         1       10        20        30                            20                            30   ProAlaAlaAlaSerHisProLeuLeuLeuAsnGlyAspAlaThrValAlaGlnLysAsn   CCAGCTGCTGCATCCCACCCTCTATTACTGAATGGAGATGCTACTGTGGCCCAGAAAAAT     40        50      60          70        80        90                            40                            50   ProGlySerValAlaGluAsnAsnLeuCysSerGlnTyrGluGluLysValArgProCys   CCAGGCTCGGTGGCTGAGAACAACCTGTGCAGCCAGTATGAGGAGAAGGTGCGCCCCTGC    100       110       120           130   140       150   IleAspLeuIleAsp    ATCGACCTCATTGAC-Z²     160       170

wherein Z³ is a 5′-end DNA residue of one or more nucleotides and Z² hasthe meaning given under formula I, in particular the cDNA of the formula

Z⁴-TGGACACGCCTCCCTCGCGCCCTTGCCGCXCACCTGCTCACCCAGCTCAGGGXCTTTGGA (III),(SEQ ID NO. 7 and 8)       -270      -260      -250      -240      -230      -220   ATTCTXTGGCCACACTGCGAGGAGATCGGTTCTGGGTCGGAGGCTACAGGAAGACTCCCA       -200      -190      -180      -170      -160      -150   CTCCCTGAAATCTGGAGTGAAGAACGCCGCCATCCAGCCACCATTCCAAGGAGGTGCAGG       -140      -130      -120      -110      -100       -90   AGAACAGCTCTGTGATACCATTTAACTTGTTGACATTACTTTTATTTGAAGGAACGTATA       -80        -70       -60       -50       -40       -30                                  1                          10                              MetValValSerGluValAspIleAlaLysAla   TTAGACCTTACTTTGCAAAGAAGGAAGATGGTTGTTTCCGAAGTGGACATCGCAAAAGCT        -20       -10         1       10        20        30                               20                            30   AspProAlaAlaAlaSerHisProLeuLeuLeuAsnGlyAspAlaThrValAlaGlnLys   GATCCAGCTGCTGCATCCCACCCTCTATTACTGAATGGAGATGCTACTGTGGCCCAGAAA        40        50        60        70        80        90                               40                            50   AsnProGlySerValAlaGluAsnAsnLeuCysSerGlnTyrGluGluLysValArgPro   AATCCAGGCTCGGTGGCTGAGAACAACCTGTGCAGCCAGTATGAGGAGAAGGTGCGCCCC       100       110       120       130       140       150                               60                            70   CysIleAspLeuIleAspSerLeuArgAlaLeuGlyValGluGlnAspLeuAlaLeuPro   TGCATCGACCTCATTGACTCCCTGCGGGCTCTAGGTGTGGAGCAGGACCTGGCCCTGCCA       160       170       180       190       200       210                               80                            90   AlaIleAlaValIleGlyAspGlnSerSerGlyLysSerSerValLeuGluAlaLeuSer   GCCATCGCCGTCATCGGGGACCAGAGCTCGGGCAAGAGCTCCGTGTTGGAGGCACTGTCA       220       230       240       250       260       270                              100                           110   GlyValAlaLeuProArgGlySerGlylleValThrArgCysProLeuValLeuLysLeu   GGAGTTGCCCTTCCCAGAGGCAGCGGGATCGTGACCAGATGCCCGCTGGTGCTGAAACTG       280       290       300       310       320       330                              120   LysLysLeuValAsnGluAspLysTrpArgGlyLysVal   AAGAAACTTGTGAACGAAGATAAGTGGAGAGGCAAGGTCAG-Z⁵       340       350       360       370

wherein Z⁴ is a 5′-end DNA residue of one or more nucleotides and Z⁵ isa DNA residue of 1500 or more coding nucleotides, a stop codon andoptionally non-coding nucleotides at the 3′-end.

Furthermore, the invention relates also to a DNA which hybridizes with aDNA of formula I, II or III, e.g. the 20-mer oligonucleotide of theformula

5′-GCTTTTGCGATGTCCACTTC-3′  (IV), (SEQ ID NO.9)

the 17-mer oligonucleotide of the formula

5′-CAGCCACCATTCCAAGG-3′  (V), (SEQ ID NO.10)

and the 21-mer oligonucleotide of the formula

5′-CGCACCTTCTCCTCATACTGG-3′  (VI), (SEQ ID NO.11)

The invention relates also to an RNA coding for proteins as describedhereinbefore, in particular to an RNA of the formula I, II or III,wherein Z¹ to Z⁵ have the meanings given hereinbefore except that RNAresidues replace DNA residues and hence uridine (U) replacesdeoxy-thymidine (T).

The DNAs coding for the desired proteins can be prepared, for example,by culturing a transformed host and isolating the desired DNA therefrom.

In particular, such DNAs can be prepared by

a) isolating mRNA from human cells, selecting the desired mRNA,preparing single-stranded DNA complementary to that mRNA, thendouble-stranded DNA (ds cDNA) therefrom, or

b) isolating genomic DNA from human cells and selecting the desired DNAusing a DNA probe, and

c) incorporating ds cDNA of step a) or ds DNA of step b) into anappropriate expression vector,

d) transforming an appropriate host microorganism with the obtainedhybrid vector,

e) selecting the transformed host which contains DNA coding for thedesired protein from hosts containing no coding DNA, and

f) isolating the desired DNA.

Polyadenylated messenger RNA is isolated from human cells by knownmethods. Suitable cells are normal lymphocytes, macrophages, monocytes,lymphoblastoid cells, e.g. Namalwa cells, human embryonic foreskindiploid cells or the like, induced with natural or recombinantinterferon o or B. Isolation methods involve, for example, lysingstimulated cells in the presence of a detergent and optionally aribonuclease inhibitor, e.g. heparin, guanidinium isothiocyanate andmercaptoethanol, extracting the mRNA with phenol or suitablechloroform-phenol mixtures, optionally in the presence of salt andbuffer solutions, detergents, proteinase and/or cation chelating agents,and precipitating mRNA from the remaining aqueous, salt-containing phasewith ethanol, isopropanol or the like. The isolated mRNA may be furtherpurified by centrifuging in a cesium chloride gradient followed byethanol precipitation and/or by chromatographic methods, e.g. affinitychromatography, for example chromatography on oligo(dT) cellulose or onoligo(U) sepharose. Preferably, crude or purified total mRNA isfractionated according to size by gradient centrifugation, e.g. in alinear sucrose gradient, or chromatography on suitable sizefractionation columns, e.g. on agarose gels.

The desired mRNA is selected by screening with a DNA probe or bytranslation in suitable cells or cell-free system and screening theobtained polypeptides. Preferably, fractionated mRNA is translated incells, e.g. in frog oocytes, or in cell-free systems, e.g. inreticulocyte lysates or wheat germ extracts. The obtained polypeptidesare compared with native protein purified as described hereinbefore,e.g. by polyacrylamide gel electrophoresis, and mRNA fractions givingrise to the desired protein selected.

The preparation of a single-stranded complementary DNA from the selectedmRNA template is well known in the art, as is the preparation of adouble-stranded DNA from a single-stranded DNA. The mRNA template isincubated with a mix of deoxynucleotide triphosphates, optionally aradioactively labelled deoxynucleotide triphosphate (in order to be ableto screen the result of the reaction), a primer sequence such as anoligo(dT) residue hybridizing with the poly(A) tail of the messenger RNAand a suitable enzyme, e.g. a reverse transcriptase. After degradationof the template mRNA, the complementary DNA (cDNA) is incubated with amix of deoxynucleotide triphosphates and a suitable enzyme as above togive a double-stranded DNA. Suitable enzymes are a reversetranscriptase, the Klenow fragment of E. coli DNA polymerase I or T₄ DNApolymerase. Optionally, the single-stranded DNA is first extended with atail of like deoxynucleotides to allow the use of a primer sequence ofcomplementary like deoxynucleotides, but the formation of dsDNA usuallystarts on spontaneous hairpin formation. Such dsDNA obtained as a resultof hairpin formation is further processed with SI nuclease which cutsthe hairpin. In a preferred alternative protocol, the mRNA/DNA hybrid istreated directly with RNase H, T₄ DNA ligase and DNA polymerase I, thusavoiding the additional steps of extending with a primer sequence and/orhairpin cutting.

As an alternative to the preparation of cDNA from mRNA, genomic DNA maybe isolated and screened for DNA coding for the desired polypeptide.

Genomic DNA is isolated from suitable human tissue, preferably fromhuman placenta or human fetal liver cells, according to methods known inthe art. A genomic DNA library is prepared therefrom by digestion withsuitable restriction endonucleases and incorporation into λ charonphage, e.g. λ charon 4A, following established procedures. The genomicDNA library replicated on nitrocellulose membranes is screened with aDNA probe, e.g. a synthetic DNA probe of at least 17 nucleotides or acDNA derived from mRNA coding for the desired polypeptide, as describedhereinbefore.

The incorporation of dsDNA prepared from mRNA or of genomic origin intoan appropriate vector is well known in the art. For example, a suitablevector is cut and provided with tails of like deoxynucleotides. ThedsDNA to be annealed then has to bear tails of complementary likedeoxynucleotides, which is accomplished by incubation in the presence ofthe corresponding deoxynucleotide triphosphate and an enzyme such asterminal deoxynucleotidyl transferase. Otherwise, the dsDNA may beincorporated into the vector by simple ligation after treatment with thesame endonuclease yielding complementary protruding ends, with the aidof linker oligonucleotides or else by blunt end ligation.

The transformation of an appropriate host microorganism with theobtained hybrid vector is well known in the art. For example, E. coliare conditioned for transformation by incubation in media containingcalcium chloride, then treated with the hybrid vector. Transformed hostsare selected by suitable markers, for example by an antibioticsresistance marker, e.g. tetracycline, chloramphenicol or ampicillinresistance, and/or by an enzyme marker, e.g. β-galactosidasecomplementing α-protein.

Hosts transformed with the desired DNA are preferably selected using aDNA probe. Such hybridization probe is e.g. a fully synthetic DNAconsisting of at least 17 nucleotides, e.g. around 20 nucleotides,constructed on the basis of the partial amino acid sequence determinedon the desired protein isolated from interferon-induced Namalwa cells.Preferably mixtures of oligonucleotide probes are prepared, wherein eachmember of the mixture is complementary to one of the possiblecombinations of triplet codons for the corresponding known amino acidsequence.

Such DNA probes are also comprised by the present invention. They aresynthesized according to known methods, preferably by stepwisecondensation using the solid phase phosphotriester, phosphite triesteror phosphoramidite method, e.g. the condensation of dinucleotidecoupling units by the phosphotriester method. These methods are adaptedto the synthesis of mixtures of the desired oligonucleotides by usingmixtures of two, three or four nucleotides dA, dC, dG and/or dT inprotected form or the corresponding dinucleotide coupling units in theappropriate condensation step as described by Y. Ike et al. [NucleicAcid Research 11, 477 (1983)].

The DNA probes have to contain a marker so that hybridization with DNAof transformed hosts can be detected and the hosts identified andseparated from other hosts not containing the desired DNA of the presentinvention. Suitable are e.g. radioactive labels such as ³²P in the5′-end phosphate of the oligonucleotide, or fluorescent markers or alabel containing biotin which can be detected with suitably labelledavidin, e.g. avidin bearing a fluorescent marker or conjugated with anenzyme such as horseradish peroxidase.

Hybridization of DNA from transformed hosts with the DNA probescontaining a marker is performed according to known procedures, i.e. inbuffer and salt solutions containing adjuncts, e.g. calcium chelators,viscosity regulating compounds, proteins, irrelevant DNA or tRNA and thelike, at temperatures favoring selective hybridization, e.g. between 0°and 70° C., for example between 40 and 50° C., preferably at around 20°lower than the hybrid dsDNA melting temperature.

The invention further relates to hybrid vectors comprising a DNA codingfor the desired proteins operatively linked to an expression controlsequence, and to processes for the preparation thereof. The vector isselected depending on the host cells envisaged for transformation.Examples of suitable hosts are microorganisms, which are devoid of orpoor in restriction enzymes or modification enzymes, such as yeasts, forexample Saccharomyces cerevisiae, for example S. cerevisiae GRF 18, andstrains of bacteria, in particular strains of Escherichia coli, forexample E. coli X1776, E. coli HB 101, E. coli W3110, E. coliHB101/LM1035, E. coli JA221, E. coli JM109 or E. coli K12 strain 294,Bacillus subtilis, Bacillus stearothermophilus, Pseudomonas,Haemophilus, Streptococcus and others, and furthermore cells of higherorganisms, in particular established human or animal cell lines, e.g.Hela cells, SV-40 virus transformed kidney cells of African green monkeyCOS-7 or chinese hamster ovary (CHO) cells. The above strains of E.coli, for example E. coli JM 109, E. coli HB101, E. coli K12 and E. coliW3110, and of Saccharomyces cerevisiae are preferred as the hostmicroorganism. In principle, all vectors which replicate and express thedesired polypeptide gene according to the invention in the chosen hostare suitable. Examples of vectors which are suitable for the expressionin an E. coli strain are bacteriophages, for example derivatives oflambda or M13 bacteriophages, or plasmids, such as, in particular, theplasmid ColEl and its derivatives, for example pMB9, pSF2124, pBR317 orpBR322. The preferred vectors of the present invention are derived fromplasmid pBR322. Suitable vectors contain a complete replicon and amarker gene, which allows to select and identify the hosts transformedwith the expression plasmids on the basis of a phenotypical trait, andoptionally signal sequences and enhancers. Suitable marker genes impartto the host, for example, resistance towards heavy metals, antibioticsand the like. Furthermore, preferred vectors of the present inventioncontain, outside the replicon and marker gene regions, recognitionsequences for restriction endonucleases, so that the gene for thedesired peptide and, if appropriate, the expression control sequence canbe inserted at these sites. The preferred vector, the plasmid pBR322 andderived plasmids, e.g. pUC9, pHRil48 and pPLc24, contain an intactreplicon, marker genes, which confer resistance e.g. towards.tetracycline and ampicillin (tet^(R) and amp^(R)), and a number ofunique recognition sites for restriction endonucleases.

Several expression control sequences can be used for regulation of thegene expression. In particular, expression control sequences of highlyexpressed genes of the host to be transformed are used. In the case ofpBR322 as the hybrid vector and E. coli as the host microorganism, forexample, the expression control sequences (which contain, inter alia,the promoter and the ribosomal binding site) of the lactose operon,tryptophan operon, arabinose operon and the like, the β-lactamase gene,the corresponding sequences of the phage λ N gene, especially thosecontaining the P_(L) promoter, or the phage fd-coat protein gene andothers are suitable. Whilst the plasmid pBR322 already contains thepromoter of the β-lactamase gene (β-lac gene), the other expressioncontrol sequences must be introduced into the plasmid.

Vectors which are suitable for replication and expression in yeastcontain a yeast replication start and a selective genetic marker foryeast. Hybrid vectors which contain a yeast replication start, forexample chromosomal autonomously replicating segment (ars), are retainedextrachromosomally within the yeast cell after the transformation andare replicated autonomously. Furthermore, hybrid vectors which containsequences homologous to the yeast 2μ plasmid DNA can be used. Suchhybrid vectors will get integrated by recombination into 2μ plasmidsalready existing within the cell, or replicate autonomously. 2μsequences are particularly suitable for plasmids with a hightransformation frequency and permit high copy numbers. The preferredyeast vector of the present invention is the plasmid pJDB207.

Suitable marker genes for yeasts are, in particular, those which impartantibiotic resistance to the host or, in the case of auxotrophic yeastmutants, genes which complement host lesions. Corresponding genesimpart, for example, resistance towards the antibiotic cycloheximide orprovide for protrophy in an auxotrophic yeast mutant, for example theURA3, LEU2, HIS3 or, in particular, TRP1 gene. Yeast hybrid vectorsfurthermore preferably contain a replication start and a marker gene fora bacterial host, in particular E. coli, so that the construction andcloning of the hybrid vectors and their intermediates can take place ina bacterial host.

Expression control sequences which are suitable for expression in yeastare, for example, those of highly expressed yeast genes. Thus, thepromoters of the TRP1 gene, the ADHI or ADHII gene, acid phosphatase(PH03 or PH05) gene, isocytochrome gene or a promoter involved with theglycolytic pathway, such as the promoter of the enolase,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglyceratekinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase andglucokinase genes, can be used. Preferred vectors of the presentinvention contain promoters with transcriptional control, e.g. thepromoters of the PH05, ADH II and CAPDH genes, which can be turned on oroff by variation of the growth conditions. For example, the PH05promoter can be repressed or derepressed solely by increasing ordecreasing the concentration of inorganic phosphate in the medium.

Vectors suitable for replication and expression in mammalian cells arepreferably provided with DNA from viral origin, e.g. from simian virus40 (SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papillomavirus (BPV), papovavirus BK mutant (BKV), or mouse or humancytomegalovirus (CMV). Preferably, such vectors contain an origin ofreplication and an antibiotics resistance gene for propagation in E.coli together with an eukaryotic transcription regulatory sequence. Inparticular, such so-called shuttle vectors may be constructed from apBR322 E. coli plasmid and SV40 and/or CMV enhancer and promoterregions. For example, the plasmid may contain the enhancer promoter unitof the mouse or human cytomegalovirus major immediate-early gene, theSV40 enhancer combined with the human α-globin promoter, and/or inaddition inducible promoters, such as the ones derived from the heatshock or metallothionein genes. Further it is also possible to utilizepromoter or control sequences which are normally associated with thedesired gene sequence. An origin of replication may be provided eitherby construction of the vector to include an exogeneous origin, such asderived from SV40, other viral source or provided by the host cellchromosomal replication mechanism. If the vector is integrated into thehost cell chromosome, the latter method is often more efficient.

In a preferred embodiment, the present invention relates to hybridvectors capable of replication and phenotypical selection in a hoststrain comprising a promoter and a DNA encoding the desired protein,said DNA being positioned together with transcription start andtermination signals as well as translation start and stop signals insaid hybrid vector under the control of said promoter such that in atransformed host it is expressed to produce the polypeptide.

The invention also relates to a process for the preparation of atransformed host, which comprises transforming or transfecting a hostwith an expression vector containing a DNA of the invention regulated byan expression control sequence, and to the transformed or transfectedhosts themselves.

Examples of suitable hosts are the above-mentioned microorganisms, suchas strains of Saccharomyces cerevisiae, Bacillus subtilis andEscherichia coli. The transformation with the expression plasmidsaccording to the invention is carried out, for example, as described inthe literature, thus for S. cerevisiae [A. Hinnen, J. B. Hicks and G. R.Fink, Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], B. subtilis[Anagnostopoulos et al., J. Bacteriol. 81, 741 (1961)] and E. coli [M.Mandel et al., J. Mol. Biol. 53, 159 (1970)].

Accordingly, the transformation procedure of E. coli cells includes Ca²⁺pretreatment of the cells so as to allow DNA uptake, and incubation withthe hybrid vector. The cells are transferred to a selective growthmedium which allows separation of the transformed cells from the parentcells. Cells which do not contain the vector will not survive in such amedium. The transformation of yeast comprises, for example, the steps of(1) enzymatic removal of the yeast cell wall by means of glucosidases,(2) treatment of the obtained spheroplasts with the vector in thepresence of polyethylene glycol and Ca²⁺ ions and (3) regeneration ofthe cell wall by embedding the spheroplasts into agar. Preferably, theregeneration agar is prepared in a way to allow regeneration andselection of the transformed cells at the same time.

Further examples of suitable hosts are the above-mentioned mammaliancells, e.g. COS-7 cells, Hela cells or chinese hamster ovary (CHO)cells. The vectors are introduced into mammalian cells by transfectionin the prescence of helper compounds, e.g. diethylaminoethyldextran,dimethyl sulfoxide, glycerol, polyethylene glycol or the like, or asco-precipitates of vector DNA and calcium phosphate. Further suitablemethods include direct microinjection of vector DNA into the cellnucleus and electroporation, i.e. introduction of DNA by a shortelectric pulse increasing the permeability of cell membranes. Thesubsequent selection of transfected cells can be done using a selectionmarker which is either covalently integrated into the expression vectoror added as a separate entity. Selection markers include genes whichconfer resistance to antibiotics, e.g. G-418 (neomycin) or hygromycin,or genes which complement a genetic Lesion of the host cell such as theabsence of thymidine kinase or hypoxanthine phosphoribosyl transferase.

The transformed host cells are cultured by methods known in the art in aliquid medium containing assimilable sources of carbon, nitrogen andinorganic salts.

Various sources of carbon can be used for culture of the transformedhosts according to the invention. Examples of preferred sources ofcarbon are assimilable carbohydrates, such as glucose, maltose, mannitolor lactose, or an acetate, which can be used either by itself or insuitable mixtures. Examples of suitable sources of nitrogen are aminoacids, such as casaminoacids, peptides and proteins and theirdegradation products, such as tryptone, peptone or meat extracts, yeastextracts, malt extract and also ammonium salts, for example ammoniumchloride, sulfate or nitrate, which can be used either by themselves orin suitable mixtures. Inorganic salts which can also be used are, forexample, sulfates, chlorides, phosphates and carbonates of sodium,potassium, magnesium and calcium.

The medium furthermore contains, for example, growth-promotingsubstances, such as trace elements, for example iron, zinc, manganeseand the like, and preferably substances which exert a selection pressureand prevent the growth of cells which have lost the expression plasmid.Thus, for example, ampicillin is added to the medium if the expressionplasmid contains an amp^(R) gene. Such an addition of antibioticsubstances also has the effect that contaminating antibiotic-sensitivemicroorganisms are destroyed. If a yeast strain which is auxotrophic in,for example, an essential amino acid is used as the host microorganism,the plasmid preferably contains a gene coding for an enzyme whichcomplements the host defect. Cultivation of the yeast strain isperformed in a minimal medium deficient in said amino acid.

Vertebrate cells are grown under tissue culture conditions usingcommercially available media optionally supplemented withgrowth-promoting substances and/or mammal sera. The cells are growneither attached to a solid support, e.g. a microcarrier or porous glassfibers, or free-floating in appropriate culture vessels.

Culturing is effected by processes which are known in the art. Theculture conditions, such as temperature, pH value of the medium andfermentation time, are chosen so that a maximum titre of the polypeptideof the invention is obtained. Thus, an E. coli or yeast strain ispreferably cultured under aerobic conditions by submerged culture withshaking or stirring at a temperature of about 20 to 40° C., preferablyabout 30° C., and a pH value of 4 to 8, preferably at about pH 7, forabout 4 to 30 hours, preferably until maximum yields of the polypeptideof the invention are reached.

When the cell density has reached a sufficient value, the culture isinterrupted and the polypeptide is isolated. If the polypeptide is fusedwith a suitable signal peptide sequence, it is excreted by the celldirectly into the supernatant. Otherwise, the cells have to bedestroyed, for example by treatment with a detergent, such as SDS,NP-40, Triton® or deoxycholic acid, or lysed with lysozyme, a similarlyacting enzyme or with ultra-sound. If yeast is used as a hostmicroorganism, the cell wall may be removed by enzymatic digestion witha glucosidase. Alternatively or additionally, mechanical forces, such asshearing forces (for example X-press, French press, Dyno mill) orshaking with glass beads or aluminium oxide, or alternating freezing,for example in liquid nitrogen, and thawing, for example to 30° to 40°C., as well as ultra-sound can be used to break the cells.

The cell supernatant or the solution obtained after centrifugation ofthe mixture obtained on breaking the cells, which contains proteins,nucleic acids and other cell constituents, is enriched in proteins,including the polypeptides of the invention, in a manner which is knownper se. Thus, for example, most of the non-protein constituents areremoved by polyethyleneimine treatment and the proteins including thepolypeptides of the invention are precipitated, for example, bysaturation of the solution with ammonium sulfate or with other salts.Otherwise, the cell supernatant or lysate is directly pre-purified usingchromatographic methods.

The polypeptides of the invention are purified by a combination ofchromatographic separations, preferably by a combination of ion exchangechromatography, gel filtration and reversed phase high performanceliquid chromatography. Other separation methods may be included in thepurification protocol, e.g. filtration or ultrafiltration with molecularweight cut-off membranes, affinity chromatography, chromatography onhydroxylapatite, chromatofocusing, and methods of dialyzing, dissolvingand reprecipitating in suitable salt and/or buffer solutions and solventmixtures.

A suitable carrier material for ion exchange chromatography may be oforganic or inorganic origin, e.g. cross-linked agarose, dextran,polyacrylamide, styrene/divinylbenzene copolymer, cellulose, or thelike. Preferably this carrier material bears basic functional groups,e.g. tertiary amino functions, quaternary ammonium groups, or slightlyacidic groups, e.g. carboxymethyl functions. The carriers may besuitable for normal liquid chromatography, fast protein liquidchromatography (FPLC) or high performance liquid chromatography (HPLC).The separations and purifications with ion exchange chromatography areperformed following established procedures, e.g. in aqueous buffersolutions of pH 4 to pH 9 containing increasing amounts of salt, forexample sodium chloride.

Carrier material suitable for gel filtration or size exclusionchromatography includes cross-linked dextran, agarose, suitably modifiedpolyacrylamide or silica, and the like. Optionally these carriers aremodified with substituents bearing hydroxy functions, e.g. with1-hydroxy- or 1,2-dihydroxy-lower alkyl groups. The chromatographicmaterial is chosen so as to display optimal separation of peptides inthe range of 50,000 to 100,000 Dalton molecular weight. Such gelfiltration or size exclusion chromatography may be performed in columnssuitable for normal liquid chromatography, FPLC or HPLC as above usingaqueous buffer solutions around neutrality containing variable amountsof salt, e.g. sodium chloride.

Reversed phase chromatography is performed on silica-based carriermaterial bearing hydrophobic groups, e.g. alkyl groups of l to 20 carbonatoms, preferably 4, 8, 12 or 18 carbon atoms or mixtures of alkylgroups of 1 and 8 or 2 and 18 carbon atoms, respectively, or phenylgroups. Related to this method is the hydrophobic interactionchromatography, wherein agarose or a related material coated with alkylgroups of up to 12 carbon atoms and/or phenyl groups is used. Thesechromatographic techniques are applied using FPLC or HPLC. Solvents forprocessing of the polypeptides of the invention on silica-based reversedphase material are aqueous acids, e.g. aqueous trifluoracetic acid,containing increasing amounts of a polar, water-miscible organicsolvent, e.g. acetonitrile, lower alcohols, e.g. methanol, ethanol orpropanol, tetrahydrofuran, and the like, preferably acetonitrile.

Affinity chromatography is also contemplated for the purification of thepeptides of the invention, using a suitable carrier material, e.g.cross-linked agarose, dextran or polyacrylamide bearing molecules withhigh affinity for the desired proteins, for example antibodies, inparticular monoclonal antibodies as described hereinbelow. Theantibodies are then coupled to the carrier material in activated form byknown methods. The purification of the desired proteins by affinitychromatography is performed in a manner known per se, e.g. in buffersolutions in a pH range of from approximately pH 5 to approximately pH 9and/or salt solutions, for example NaCl solution, optionally containingsurfactants, e.g. polyethylenesorbitan fatly acid esters, then elutingthe desired proteins with buffer solutions in a pH range of fromapproximately pH 2 to approximately pH 5, such as glycine buffer, or pHgradients of differing composition or salt solutions, for exampleconcentrated NH₄ SCN solution.

The antiviral properties of the proteins of the invention are useful forthe therapy of and/or protection against viral infections. Inparticular, the proteins may be used for treating influenza and otherrespiratory tract virus infections, herpes virus infections, and rabiesand hepatitis infection, optionally in combination with other antiviralagents. The proteins are applied in the form of pharmaceuticalpreparations that contain a therapeutically effective amount of theactive ingredient optionally together or in admixture with inorganic ororganic, solid or liquid, pharmaceutically acceptable carriers.

The pharmaceutical preparations according to the invention are those forenteral, e.g. rectal or oral, administration and preferably forparenteral, e.g. intranasal, intramuscular, subcutaneous or intravenous,administration to warm-blooded animals, for example humans.

Depending on the intended method of administration, the pharmaceuticalpreparations may be in unit dose form, for example in ampoules, vials,suppositories, dragees, tablets, capsules or nasal sprays in liquid orsolid form.

The amount of the therapeutically effective compounds to be administereddepends on the condition of the warm-blooded animal, for example thehuman, such as the body weight, the nature and severity of the diseaseand the general condition and also on the mode of administration, and iscarried out in accordance with the assessment of the physician givingthe treatment. The effective dose is in the order of magnitude of from0.001 to 1 μg per kg of body weight per day.

The pharmaceutical preparations according to the invention contain thecustomary inorganic or organic, solid or liquid pharmaceuticallyacceptable carriers, optionally together with other therapeuticallyeffective compounds and/or adjuncts. There are preferably used solutionsor suspensions of the active ingredient, especially isotonic aqueoussolutions or suspensions, or also lyophilized preparations which aredissolved in water shortly before use. The pharmaceutical preparationsmay be sterilized and/or contain preservatives, stabilizers, wettingagents, emulsifiers, solubilizers, viscosity-increasing substances,salts for regulating the osmotic pressure and/or buffers, and also otherproteins, for example human serum albumin or human blood plasmapreparations.

Further, the invention relates to monoclonal antibodies specific to thehuman proteins induced by interferon α or β as described hereinbefore,particularly to monoclonal antibodies which do not crossreact with therelated mouse protein Mx induced by interferon, and to derivatives ofsuch antibodies.

The monoclonal antibodies of the invention are preferably of murineorigin, and are particularly mouse antibodies produced by mouse/mousehybridoma cells.

Examples of monoclonal antibodies of the invention are the mousemonoclonal antibodies with the designation 885 S35.8.1, 885 S35.16.11,885 S56.55.7.12.48, 885 S56.55.7.21.25, 885 S56.55.7.27.5, 885S56.55.7.27.11, 885 S56.55.13, 885 S56.55.17, and 885 S56.67.15.

Preferred are the monoclonal antibodies with the designation 885S35.8.1, 885 S56.55.13 and 885 S56.67.15, and derivatives thereof. Thesemonoclonal antibodies are secreted by the corresponding hybridoma celllines with the designation 885 S35.8.1, 885 S56.55.13 and 885 S56.67.15.

Derivatives of monoclonal antibodies of this invention are e.g. antibodyfragments, radioactively labelled monoclonal antibodies, and conjugatesof the monoclonal antibodies with enzymes, with fluorescent markers, orthe like.

Fragments of monoclonal antibodies of this invention are e.g. Fab, Fab′or F(ab′)₂ fragments, which retain their specificity for the antigenicdeterminants, i.e. which retain the specificity for the humaninterferon-induced proteins as described hereinbefore.

Radioactively labelled monoclonal antibodies contain e.g. radioactiveiodine (¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H) orthe like. Preferred are monoclonal antibodies labelled with radioactiveiodine.

Antibody conjugates of the invention are e.g. conjugates of monoclonalantibodies or fragments thereof with enzymes such as horseradishperoxidase, alkaline phosphatase, B α-D-galactosidase, glucoseoxidase,glucoamylase, carboanhydrase, acetylcholinesterase, lysozyme, malatedehydrogenase or glucose-6-phosphate dehydrogenase, with fluorescentmarkers, e.g. fluorescein, or with avidin or biotin. In such conjugatesthe antibody is bound to the enzymes or fluorescent marker directly orby the way of a spacer or linker group. Preferred are conjugates ofmonoclonal antibodies with the enzymes horseradish peroxidase oralkaline phosphatase.

The monoclonal antibodies of the invention and derivatives thereof areobtained by processes known per se, characterized in that hybridomacells secreting said monoclonal antibodies

a) are cultivated in vitro and the monoclonal antibodies isolated fromthe culture supernatant, or

b) are propagated in vivo in a suitable mammal and the monoclonalantibodies recovered from body fluids of said mammal, and, if desired,

c) the obtained monoclonal antibodies are converted into a derivativethereof.

Suitable culture media for the in vitro cultivation according to processa) are standard culture media such as Dulbecco's Modified Eagle Mediumor RPMI 1640 Medium, optionally replenished by a mammal serum, e.g.fetal calf serum, or other growth-sustaining supplements, e.g.2-aminoethanol, insulin, transferrin, low density lipoprotein, oleicacid and the like, and trace elements. The isolation of the monoclonalantibodies is accomplished by precipitating the protein contained in theculture supernatants by ammonium sulfate or the like, followed bypurifying the immunoglobulins by standard chromatographic methods, suchas gel filtration, ion exchange chromatography, chromatography on DEAEcellulose, or immunoaffinity chromatography.

In vitro production allows scale-up to give large amounts of the desiredantibodies. Techniques for large scale hybridoma cultivation are knownin the art and include homogeneous suspension culture, e.g. in anairlift reactor or in a continuous stirrer reactor, or immobilized orentrapped cell culture, e.g. in hollow fibers, microcapsules, on agarosemicrobeads or ceramic cartridges.

Large amount s of the desired mono clonal antibodies can also beobtained by the propagation of hybridoma cells according to process b).Cell clones are injected into syngeneic mammals, which causesantibody-producing tumors to grow. After one to three weeks the desiredmonoclonal antibodies are recovered from body fluids of said mammal. Asan example hybridoma cells derived from Balb/c mice areintraperitoneally injected into Balb/c mice optionally pretreated with ahydrocarbon such as pristane, and after one to two weeks, ascites fluidof these mice is collected. The desired monoclonal antibodies areisolated from the body fluids by methods known per se, e.g. byprecipitating the proteins with ammonium sulfate or the like, followedby purifying the immunoglobulins by standard chromatographic methods,such as gel filtration, ion exchange chromatography, chromatography onDEAE cellulose, or immunoaffinity chromatography.

Fragments of monoclonal antibodies, for example Fab, Fab′ or F(ab′)₂fragments, which retain their specificity towards the humaninterferon-induced proteins as described hereinbefore, can be obtainedfrom the monoclonal antibodies pre pared according to process a) or b)by methods known per se, e.g. by digestion with enzymes such as pepsinor papain and/or cleavage of disulfide bonds by chemical reduction.

Monoclonal antibodies labelled with radioactive iodine are prepared byiodination methods known in the art, e.g. by labelling monoclonalantibodies with radioactive sodium or potassium iodide and a chemicaloxidant, such as sodium hypochlorite, chloramine T or the like, or anenzymatic oxidant, such as lactoperoxidase or glucose oxidase andglucose. Radioactively labelled monoclonal antibodies o f the inventionare also prepared by adding radioactively labelled nutrients to theculture media of the in vitro cultivation of step a). Such labellednutrients contain e.g. radioactive carbon (¹⁴C), tritium (³H), sulfur(³⁵S) or the like, and are for example L-(¹⁴C)-leucine, L-(³H)-leucineor L-(³⁵S)-methionine.

Conjugates of monoclonal antibodies of the invention are prepared bymethods known in the art, e.g. by reacting a monoclonal antibodyprepared according to process a) or b) or a fragment thereof prepared asdescribed hereinbefore with the enzyme in the presence of a couplingagent, e.g. glutaraldehyde, periodate, N,N′-o-phenylenedimaleimide,N-(m-maleimidobenzoyloxy)-succinimide,N-(3-[2′-pyridyldithio]-propionoxy)-succinimide,N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide or the like. Conjugateswith avidin are prepared likewise. Conjugates with biotin are preparede.g. by reacting monoclonal antibodies with an activated ester of biotinsuch as the biotin N-hydroxysuccinimide ester. Conjugates withfluorescent markers are prepared in the presence of a coupling agent,e.g. those listed above, or by reaction with an isothiocyanate,preferably fluorescein-isothiocyanate.

The invention further relates to hybridoma cell lines, characterized inthat they secrete monoclonal antibodies with specificity for the humaninterferon-induced proteins as described hereinbefore.

In particular, the invention relates to cell lines, which are hybrids ofmyeloma cells and B lymphocytes of a mammal immunized with purifiedhuman interferon-induced protein with apparent molecular weight of 78kDa. Preferentially, these cell lines are hybrids of mouse myeloma cellsand B lymphocytes of a syngeneic mouse immunized with the protein.

Examples of such cell lines are the hybridoma cell lines with thedesignation 885 S35.8.1, 885 S35.16.11, 885 S56.55.7.12.48, 885S56.55.7.21.25, 885 S56.55.7.27.5, 885 S56.55.7.27.11, 885 S56.55.13,885 S56.55.17, and 885 S56.67.15.

These hybridoma cell lines are hybrids of the mouse myeloma cell lineSp2/0-Ag14 and of B lymphocytes of the spleen of Balb/c mice immunizedwith the purified human interferon-induced protein from Namalwa cells asdescribed hereinbefore. They are stable cell lines and secrete themonoclonal antibodies with the corresponding designation. The cell linesmay be kept in culture or deep-frozen in liquid nitrogen and reactivatedby thawing.

Particularly preferred are the hybridoma cell lines with the designation885 S35.8.1, 885 S56.55.13 and 885 S56.67.15, which have been depositedon Apr. 9, 1986 at the “Collection Nationale de Cultures deMicroorganismes”, Institut Pasteur, Paris, under the number I-545,I-543, and I-544, respectively.

The invention relates also to a process for the production of hybridomacell lines secreting monoclonal antibodies with specificity for theinterferon-induced proteins as described hereinbefore, characterized inthat a suitable mammal is immunized with a purified protein, optionallywith an antigenic carrier, antibody-producing cells of this mammal arefused with myeloma cells, the hybrid cells obtained in the fusion arecloned, and cell clones secreting the desired antibodies are selected.

Preferred mammals for the immunization are mice, particularly HR-mice.The immunizations are performed e.g. by implanting an antigenic carrier,e.g. a nitrocellulose piece, containing purified 78 kDa protein frominduced Namalwa cells, and further injecting between 2 μg and 10 μg ofthe protein two to ten times parenterally, such as intraperitoneallyand/or subcutaneously, at intervals of 7 to 30 days. The injectionsoptionally contain an adjuvant stimulating the lymphocyte productionsuch as complete or incomplete Freund's adjuvant and/or an adjuvantpeptide.

Antibody-producing cells of the immunized mammals, preferably spleencells, taken two to five days after the final booster injection, arefused with myeloma cells of a suitable cell line in the presence of afusion promoter. Several suitable myeloma cell lines are known in theart. Preferred are myeloma cell lines lacking the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT) or the enzyme thymidinekinase (TK), which therefore do not survive in a selective culturemedium containing hypoxanthine, aminopterin and thymidine (HAT medium).Particularly preferred are myeloma cells and derived cell lines that donot survive in HAT medium and do not secrete immunoglobulins orfragments thereof, such as the cell lines X63-Ag8.653 or Sp2/0-Ag14.Fusion promoters considered are e.g. Sendai virus or other paramyxoviruses, optionally in UV-inactivated form, calcium ions, surface-activelipids such as lysolecithin, or polyethylene glycol. Preferentially, themyeloma cells are fused with a three- to twentyfold excess of spleencells from immunized mammals in a solution containing about 30% to about60% polyethylene glycol of a molecular weight between 1000 and 4000.

After the fusion, the cells are resuspended and cultivated in selectiveHAT medium. Thereby, only hybridoma cells will survive, because theycombine the ability to grow and replicate in vitro, which ability isinherited from myeloma cells, with the missing HGPRT or TK genesessential for the survival in the HAT medium, which genes are inheritedfrom the antibody-producing spleen cells of the immunized mammals.

Suitable culture media for the expansion of hybridoma cells are thestandard culture media, such as Dulbecco's Modified Eagle Medium,minimum essential medium, RPMI 1640 medium and the like, optionallyreplenished by serum, e.g. 10 to 15% fetal calf serum. Preferentiallyfeeder cells are added at the beginning of the cell growth, e.g. normalmouse peritoneal exsudate cells, spleen cells, marrow bone macrophages,or the like. The culture media are supplemented with selective HATmedium at regular interval in order to prevent normal myeloma cellsovergrowing the hybridoma cells.

The hybridoma cell culture supernatants are screened for the desiredmonoclonal antibodies, preferentially with an enzyme immunoassay, e.g. adot-ELISA assay, or a radioimmunoassay. Positive hybridoma cells arecloned, e.g. by limiting dilution, preferentially twice or more.Optionally, hybridoma cells are passaged through animals, e.g. mice, byi.p. injection and harvesting of ascites, which stabilizes hybridomasand improves growth characteristics. The cloned cell lines may be frozenin a conventional manner.

The monoclonal antibodies of the invention and/or their derivatives areuseful for the qualitative and quantitative determination of theinterferon-induced human proteins as described hereinbefore.

For instance, the monoclonal antibodies or derivatives thereof, such asenzyme conjugates or radioactive derivatives, can be used in any of theknown immunoassays, which rely on the binding interaction between theantigenic determinant of the proteins of the invention and themonoclonal antibodies. Examples of such assays are radioimmunoassays(RIA), enzyme immunoassays, e.g. enzyme-linked immunoadsorbent assay(ELISA), immunofluorescence, immunoprecipitation, latex agglutination,and hemagglutination. Such immunoassays are useful e.g. in themonitoring of the production and purification of the desired proteinsfrom natural sources or genetically engineered microorganisms and in thequalitative and quantitative determination of the proteins in biologicalfluids, e.g. of patients under therapy with a protein of the inventionor with interferon, or in need of such therapy.

The monoclonal antibodies according to the invention can be used as suchor in the form of radioactively labelled derivatives in aradioimmunoassay (RIA). Any of the known modifications of an RIA can beused, for example RIA in homogeneous phase, solid phase RIA orheterogeneous RIA, single RIA or double (sandwich) RIA with direct orindirect (competitive) determination of the protein of the invention.There is preferred a sandwich RIA in which a suitable carrier, forexample the plastics surface of a microtitre plate or of a test tube,for example of polystyrene, polypropylene or polyvinyl chloride, glassor plastics beads, filter paper, or dextran, cellulose acetate ornitrocellulose sheets or the like, is coated with a monoclonal antibodyof the invention by simple adsorption or optionally after activation ofthe carrier, for example with glutaraldehyde or cyanogen bromide, andincubated with the test solution and a solution of a monoclonal antibodyradioactively labelled with ¹²⁵I, the dissolved monoclonal antibodyrecognising another epitope of the proteins of the invention than thecarrier-bound monoclonal antibody, and the amount of the proteins of theinvention is determined by measuring the radioactivity bound to thecarrier.

Particularly preferred is a sandwich radioimmunoassay as describedhereinbefore, wherein a monoclonal antibody of the invention is bound toa bead, for example a polystyrene bead, this coated bead is incubated ina test or standard solution containing interferon induced human proteinsand is finally developed with a radiolabelled monoclonal antibodyrecognizing a different epitope.

The monoclonal antibodies according to the invention can be used as suchor in the form of enzyme-conjugated derivatives in anenzyme-immunoassay. Such immunoassays include test procedures in whichenzyme-labelled monoclonal antibody derivatives according to theinvention or enzyme-labelled antibodies known per se that recognize andbind an epitope of the antibodies according to the invention are used.

There is preferred an ELISA (enzyme-linked immunoadsorbent assay) inwhich a carrier as described above for an RIA is coated with amonoclonal antibody according to the invention, incubated with a testsolution containing an interferon-induced human protein and then with apolyclonal serum to the protein, for example sheep serum, and, finally,the bound antibodies of the polyclonal serum are developed byenzyme-labelled antibodies that recognize and bind to them, and theamount of the protein bound is determined by an enzyme substratereaction. Such an enzyme-labelled antibody is, for example, aphosphatase-labelled goat-anti-sheep immunoglobulin.

There is also preferred an ELISA in which a carrier coated with amonoclonal antibody according to the invention is incubated with a testsolution and with a solution of a monoclonal antibody that is conjugatedwith an enzyme, the dissolved monoclonal antibody recognizing adifferent epitope of the interferon-induced human protein than does thecarrier-bound monoclonal antibody. By an enzyme substrate reaction thatresults, for example, in a color change and can be observed by eye orwith optical measuring devices, the amount of bound enzyme, which isproportional to the amount of the protein in the test solution, ismeasured.

Particularly preferred is an enzyme immunoassay called immunodotanalysis, in which test or standard solutions containing theinterferon-induced human protein are spotted on a microporous carrierwith high intrinsic affinity for polypeptides, e.g. on nitrocellulose,the carrier bearing one or several dots of said samples is incubated ina solution of a monoclonal antibody of the invention, then in a solutionof an enzyme-labelled second antibody that recognizes and binds themonoclonal antibody of the invention and finally in a solution of anenzyme substrate which leads to a detectable signal, e.g. a coloredsubstance. Such an enzyme-labelled second antibody is e.g. rabbitanti-mouse immunoglobulin conjugated with horseradish peroxidase whichcan be developed with suitable enzyme substrates such as4-chloro-1-naphthol or the like.

The monoclonal antibodies according to the invention can be used as suchor in the form of derivatives conjugated with fluorescent markers inimmunofluorescence tests. Such immunofluorescence tests includeprocedures wherein monoclonal antibody derivatives according to theinvention, e.g. derivatives conjugated with fluorescein, or fluorescentmarker-labelled antibodies known per se that recognize and bind anepitope of the monoclonal antibodies according to the invention areused.

There is preferred an immunofluorescence test in which a carrier asdescribed above for an RIA is coated according to standard methods withcells to be tested for the presence of a protein of the invention, thecells are fixed and permeabilized to allow interaction of proteinaceousmaterial inside the cell with solutions applied, then incubated with asolution of a monoclonal antibody derivative according to the inventionconjugated with a fluorescent marker, or incubated with a solution of amonoclonal antibody of the invention followed by a solution of afluorescent marker-labelled second antibody that recognizes and bindsthe monoclonal antibody of the invention, e.g. a fluorescein-labelledrabbit anti-mouse immunoglobulin. The presence of a protein of theinvention is then detected and the protein localized by standardfluorescence microscopy or flow cytometry.

The monoclonal antibodies according to the invention can be used as suchor in the form of radiolabelled derivatives in immunoprecipitationtests. Preferred is an immunoprecipitation test wherein cells to betested for their ability to produce a protein of the invention are grownin culture media containing radioactively labelled nutrients, e.g.nutrients labelled with radioactive carbon (¹⁴C), tritium (³H), sulfur(³⁵S) or the like, for example (³⁵S)-methionine, then lysed to obtain asolution of radiolabelled proteinaceous material produced by the cells.This solution is incubated with a solution of a monoclonal antibody ofthe invention, any complex between radiolabelled protein formed in thecell and the monoclonal antibody of the invention precipitated or,preferably, adsorbed on affinity chromatography material with highaffinity for the monoclonal antibodies of the invention, e.g.chromatography material coupled to protein A or to an antibodyrecognizing and binding the monoclonal antibodies of the invention, e.g.to rabbit anti-mouse immunoglobulin, and the protein/antibody complexisolated from the precipitate or the affinity chromatography material.The presence of the radiolabelled protein is then confirmed by usualanalytical methods, e.g. SDS polyacrylamide gel electrophoresis withfluorography, under conditions dissociating the protein/antibodycomplex.

The use according to the invention of monoclonal antibodies andderivatives thereof as described hereinbefore for the qualitative andquantitative determination of the human interferon-induced proteins alsoincludes other immunoassays known per se, for example latexagglutination with antibody-coated or antigen-coated latex particles orhemagglutination with antibody-coated or antigen-coated red bloodcorpuscles or the like.

The invention relates also to test kits for the qualitative andquantitative determination of human interferon-induced proteins withapparent molecular weight of 78 kDa containing monoclonal antibodies ofthe invention and/or derivatives thereof and, optionally, othermonoclonal or polyclonal antibodies and/or adjuncts.

Test kits according to the invention for a radioimmunoassay contain, forexample, a suitable carrier, uncoated or coated with a monoclonalantibody of the invention, optionally freeze-dried or concentratedsolutions of a monoclonal or polyclonal antibody to a protein of theinvention and/or a radiolabelled derivative thereof, standard solutionsof this protein, buffer solutions and, optionally, polypeptides anddetergents for preventing non-specific adsorption and aggregateformation, pipettes, reaction vessels, calibration curves, instructionmanuals and the like.

Test kits according to the invention for an enzyme immunoassay contain,for example, a suitable carrier, e.g. microtiter plates ornitrocellulose sheets, optionally freeze-dried or concentrated solutionsof a monoclonal antibody to a protein of the invention and of anenzyme-labelled monoclonal or polyclonal antibody to this protein or toa first antibody recognizing the protein, enzyme substrates in solid ordissolved form, standard solutions of a protein of the invention, buffersolutions and, optionally, polypeptides and detergents, pipettes,reaction vessels, calibration curves, color scale tables, instructionmanuals and the like.

Test kits according to the invention for an immunofluorescence testcontain, for example, a suitable carrier, e.g. plastic coverslips orglass slides, optionally freeze-dried or concentrated solutions of amonoclonal antibody to a protein of the invention and of afluorescein-labelled polyclonal antibody recognizing the monoclonalantibody, buffer solutions and, optionally, standard solutionscontaining a protein of the invention, polypeptides and detergents,pipettes, reaction vessels, instruction manuals and the like.

Test kits according to the invention for an immunoprecipitation testcontain, for example, a suitable carrier, e.g. plastic or glass plates,optionally freeze-dried or concentrated solutions of a monoclonalantibody to a protein of the invention, solutions of radiolabellednutrients, e.g. ³⁵S-methionine, tissue culture solutions, buffersolutions, optionally freeze-dried or concentrated solutions of aninterferon α or β, and, optionally, standard solutions containing aprotein of the invention, affinity chromatography material binding themonoclonal antibody in an antigen/antibody complex, detergents andpolypeptides, pipettes, reaction vessels, instruction manuals and thelike.

The monoclonal antibodies and antibody derivatives of the invention areused for the qualitative and quantitative determination of the human 78kDa protein induced by interferon α or β, preferably in enzymeimmunoassays, immunofluorescence tests or immunoprecipitation tests. Thereliable determination of the amount of human 78 kDa in biologicalfluids, tissue sections and cells allows a simple surveillance of atherapy with the human 78 kDa protein or of a therapy with interferon αor β. Furthermore, the monoclonal antibodies and antibody derivativescan be used in the isolation and purification of human 78 kDa proteinfrom natural sources or from recombinant host cells by immunoaffinitychromatography.

The following examples illustrate the invention, but do not limit it toany extent.

The abbreviations used in the examples have the following meaning:

ATP adenosine triphosphate BSA bovine serum albumin cDNA complementaryDNA cpm counts per min (radioactive decay) dA 2′-deoxyadenosine dATP2′-deoxyadenosine triphosphate dC 2′-deoxycytidine dCTP 2′-deoxycytidinetriphosphate dG 2′-deoxyguanosine dGTP 2′-deoxyguanosine triphosphateDNA deoxyribonucleic acid dNTP mixture of dATP, dCTP, dGTP and dTTP dsDNA double-stranded DNA dT (2′-deoxy-)thymidine dTTP thymidinetriphosphate EDTA ethylenediamine-tetraacetic acid FCS foetal calf serumHAT hypoxanthine/aminopterin/thymidine IFN interferon kDa kilo-Dalton(molecular weight) mRNA messenger RNA PBS phosphate buffered saline RNAribonucleic acid rpm revolutions per min SDS sodium dodecyl sulfate TBSTris buffered saline Tris tris(hydroxymethyl)aminomethane tRNA transferRNA

The following buffer solutions and media are used:

Denhardt solution 0.1% polyvinylpyrrolidone (PVP-360, Sigma), 0.1%Ficoll 400 (Pharmacia), 0.1% BSA. hypotonic buffer 5 mM Tris.HCl, pH7.4, 1.5 mM KCl, 2.5 mM MgCl₂. LB medium 1% Bacto ® tryptone (Difco),0.5% Bacto ® yeast extract (Difco), 170 mM NaCl, adjusted to pH 7.5 withNaOH. ligation buffer 50 mM Tris HCl, pH 8, 7 mM MgCl₂, 1 mM dithio-threitol. mung bean 30 mM NaOAc, pH 5, 50 mM NaCl, 1 mM ZnCl₂, nucleasebuffer 5% glycerol. PBS 136 mM NaCl, 2 mM KCl, 8 mM Na₂HPO₄, 1.4 mMKH₂PO₄. SSC buffer 15 mM sodium citrate, 150 mM NaCl, adjusted to pH 7.0with NaOH. TBS 10 mM Tris.HCl, pH 7.6, 0.15 M NaCl. TE buffer 10 mMTris.HCl, pH 7.5, 1 mM EDTA.

EXAMPLE 1 Induction of Namalwa Cells with Interferon

1.1 Cell Line:

Namalwa cells ATCC CRL 1432 are cultured in a medium consisting of RPMI1640 medium supplemented with 2 g/l NaHCO₃, penicillin (10⁵units/liter), streptomycin (100 mg/liter) and 10% inactivated FCS(inactivation: 30 min at 56° C.), in suspension culture in one literSpinner flasks (Bellco). The cells are seeded at a concentration of5×10⁵ cells per ml, and subcultured when the concentration reaches20×10⁵ cells per ml (about three times a week).

1.2 Incubation with Interferon Alpha:

2 liter of medium are seeded with Namalwa cells at a concentration of5×10⁵ cells per ml. They are cultured in a 3 liter Spinner flask for 3days at 37° C. At the end of the exponential growth, the concentrationof cells reaches 2 to 3×10⁶ cells per ml. The cells are centrifuged at800×g for 30 min, then resuspended in 2 liter of culture medium andincubated for 6 h at 37° C. Interferon 5₁ (α/β type prepared accordingto EP-A 76 489) is added at a final concentration of 5000 internationalunits per ml, and the cultures further incubated at 37° C. for 20 h.

1.3 Harvest of Cells:

The cells are centrifuged for 30 min at 1000×g. The cell pellet iswashed with PBS. The cells are centrifuged for 10 min at 800×g, and thepellet suspended in hypotonic buffer. The cells are centrifuged for 10min at 800×g, and the pellet is frozen rapidly on dry ice and kept at−20° C.

EXAMPLE 2 Isolation and Purification of the 78 kDa Protein

2.1 Protein Extraction:

Thawed cells of Example 1 are lysed at 20° C. with 200 ml of buffer 50mM Tris.HCl, pH 7.4, and 4 M NaCl. The lysate is clarified byultracentrifugation at 80,000×g for 1 h. The IFN-induced protein is inthe supernatant. Ammonium sulfate is slowly added to the supernatant toa final concentration of 30%. The proteins are precipitated for 1 h at20° C. The precipitate containing the IFN-induced protein is centrifugedfor 15 min at 3000×g, then suspended in 3 ml buffer containing 50 mMTris.HCl, pH 8, 150 mM mercaptoethanol, 6 M urea and 2% NP-40. Thesuspension is extensively dialysed against the same buffer. Most of theIFN-induced protein remains insoluble.

2.2 Preparative Gel Electrophoresis:

The insoluble proteins are centrifuged and dissolved in sample buffer[U. K. Laemmli & M. Favre, J. Mol. Biol. 80, 575 (1973)]. The slab gels(1.5 mm thick and 110 mm long) are prepared as described by Laemmli &Favre. The separating gel contains 12% acrylamide and 0.32%bis-acrylamide. At the end of the electrophoresis the proteins arevisualized by dipping the gel into ice-cold 0.25 mM KCl. The piece ofgel containing proteins of molecular weight between 70 kDa and 85 kDa iscut out. The gel is extensively washed with H₂O, equilibrated with 50 mMN-ethylinorpholinium acetate, pH 8.5, and 0.1% SDS. Finally the gel issliced in 2 nm urea, 50 mM N-ethylmorpholinium acetate, pH 8.5, 2% SDS,and 50 mM dithiothreitol. The mixture is incubated for 1 h at 37° C.

2.3 Electrodialysis of Proteins from the Gels:

An ISCO sample concentrator (Model 1750) is used to elute the proteinsfrom the gel pieces, as described by A. J. Brown & J. C. Bennett[Methods in Enzymology 91, 450 (1983)]. N-Ethylmorpholinium acetate, pH8.5, containing 0.01% SDS and 1 mM dithiothreitol is used as buffer inthe outer (0.1 M) and inner (0.05 M) chambers of the concentrator tank,respectively. The eluted proteins are precipitated with 5 volumes ofacetone.

2.4 Final Purification by Polyacrylamide Gel Electrophoresis in TwoDimensions:

The two-dimensional system combining non-equilibrium pH gradientelectrophoresis (NEPHGE) with SDS-polyacrylamide gel electrophoresis isused as described by P. Z. O'Farrell et al. [Cell 12, 1133 (1977)]. Theproteins of the acetone precipitate (Example 2.3) are solubilized in“lysis buffer A” [P. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975)] andapplied to the acidic end of the non-equilibrium pH gradientelectrophoresis gel, which contains 2% ampholytes, pH 3-10. Theelectrophoresis is run for 5 h at 500 V. The separating gel for slab gelelectrophoresis in the second dimension contains 12% acrylamide and0.32% bis-acrylamide. Proteins are visualized by dipping the gel intoice-cold 0.25 M KCl. The piece of gel containing the IFN-inducedprotein, a single spot free of other proteins, is cut out and processedfor electrodialysis as described above in Example 2.3. The purifiedprotein is precipitated with 5 volumes of acetone.

EXAMPLE 3 Characterization of the Purified 78 kDa Protein:

3.1 SDS Polyacrylamide Gel Electrophoresis

The purified protein is analyzed by one-dimensional gel electrophoresison 12% polyacrylamide gels in the usual way. The bands are stained withCoomassie blue G-250. The molecular weight markers (from Bio-Rad) run inparallel are: lysozyme (14 kDa), soybean trypsin inhibitor (21.5 kDa),carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin(66.2 kDa) and phosphorylase B (92.5 kDa). The purified IFN-inducedprotein is homogenous in this type of analysis and migrates as a proteinwith approximate molecular weight 78 kDa.

The isoelectric point of the IFN-induced protein is 6.3 as determined inthe system described by P. H. O'Farrell [J.Biol.Chem. 250, 4007 (1975)].

3.2 N-terminal Amino Acid Sequence

32 μg of the protein are subjected to an amino acid sequence analysis ina Beckman 8906 sequencer in the manner described by J. Y. Chang et al.[Biochem. J. 211, 173 (1983)].

The following N-terminal sequence is found:Val-Val-X₃-Glu-Val-Asp-Ile-Ala-Lys-Ala-Pro-Lys-Ala. (SEQ ID NO.12).

The third amino acid could not be identified.

3.3 Total Amino Acid Composition:

Total amino acid composition is determined following a procedure of J.Y. Chang, R. Knecht & D. G. Braun [Methods in Enzymology, Vol. 91, 41-48(1983)]. Briefly, the protein is hydrolyzed with 6 M HCl, derivatizedwith 4′-dimethylamino-azobenzene-4-sulfonyl chloride in sodiumbicarbonate buffer, and injected on a Zorbax-ODS® high performanceliquid chromatography (HPLC) column. The amount of each amino acid isdetermined by comparison with a standard sample. The results arecollected in Table 1.

EXAMPLE 4 Isolation of mRNA from Cells Induced with Interferon

4.1 Induction of Human Embryonic Foreskin Cells with Interferon Alpha:

Human embryonic foreskin diploid cells (Flow No. 7000) are cultured inEarl's minimum essential medium supplemented with NaHCO₃ (2 g/liter),penicillin (10⁵ units/liter), streptomycin (100 mg/liter) and 10%inactivated FCS (inactivation: 30 min at 56° C.) in plastic dishes of 14cm diameter. Confluent cell monolayers are subcultured in a trypsin/EDTAsolution (Gibco) at a split ratio of 1 to 3. Confluent cell monolayersare incubated in fresh medium containing recombinant interferon 5₁ (α/Btype prepared according to EP-A 76 489) at a final concentration of 1000international units per ml for 4.5 h at 37° C.

4.2 Purification of Cytoplasmic RNA:

The cell monolayer of Example 4.1 is washed with PBS at 4° C. andincubated in hypotonic buffer for 2 min at 4° C. The cytoplasmic extractis obtained by lysis of cells with the hypotonic buffer containing 1%.deoxycholate and 1% NP-40 for 5 min at 4° C. The extract is centrifugedat 25,000×g for 5 min. To the supernatant (45 ml) are added 16 mgproteinase K, 720 mg NaCl, 1.8 ml 1 M Tris.HCl, pH 7.4, and 6.8 ml 10%SDS. The mixture is kept at 20° C. for 4 h. The RNA is extracted 3 timeswith phenol saturated with a solution of 0.1 M Tris.HCl, pH 9, and 0.1%oxyquinoline. NaCl is added to the aqueous phase (final concentration0.1 M) and the RNA is precipitated with 2 volumes of ethanol at −20° C.

4.3 Further Purification of Total RNA:

2 mg RNA of Example 4.2 in 50% formamide are layered onto a linear 5 to20% sucrose gradient in 5 mM EDTA, 0.01 M Tris.HCl, pH 7.5, 0.2% SDS,0.05 M NaCl, and 50% formamide. The gradients are centrifuged at 20° C.for 16 h at 40,000 rpm in a Beckman SW41 Ti rotor. 1 ml fractions arecollected, made up to 0.1 M NaCl, and the RNA precipitated with 2volumes of ethanol. An RNA aliquot of each fraction is translated in areticulocyte lysate cell free system (Amersham International No. N90)according to the instructions of the manufacturer. Proteins synthesizedin vitro and labelled with ³⁵S-methionine are separated bypolyacrylamide gel electrophoresis in two dimensions, and detected byfluorography. mRNA directing the synthesis of an IFN-induced protein ofapparent molecular weight 78 kDa is reproducibly found in fractions 8and 9, at sedimentation values between 18S and 28S. Poly(A) mRNA offractions 8 and 9 is purified by chromatography on oligo(dT) cellulose.

EXAMPLE 5 Preparation and Screening of a cDNA Library

Starting with purified mRNA of Example 4.3, a cDNA library is preparedfollowing the method of U. Gubler and B. J. Hoffman, Gene 25, 263-269(1983) with some modifications.

For the synthesis of the first strand cDNA, the purified poly(A) mRNA offractions 8 and 9 (Example 4.3, 150 μg/ml) is incubated in a volume of20-40 μl containing 50 mM Tris.HCl, pH 8.3, 10 mM MgCl₂, 10 mMdithiothreitol, 1.25 mM of each dGTP, dATP and dTTP, 0.5 mM dCTP, 20 μCiof α-³²P-dCTP (ca. 3000 Ci/mmol) and 100 μg/ml of oligo(dT₁₂₋₁₈) with3000 units per ml of “Super” reverse transcriptase from avianmyeloblastosis virus (Anglian Biotechnology-Stehelin) for 30 min at 43°C. The reaction is stopped by adding EDTA, the products extracted withphenol and precipitated with ethanol. For second strand synthesis, thesingle-stranded cDNA (500 ng) is incubated in 100 μl of 20 mM Tris.HCl,pH 7.5, 5 mM MgCl₂, 10 mM (NH₄)₂SO₄, 100 mM KCl, 0.15 mM β-nicotinamideadenine dinucleotide, 50 μg/ml BSA and 40 mM of each dNTP with 8.5units/ml of E. coli RNase H, 230 units/ml DNA polymerase I and 10units/ml T₄ DNA ligase overnight at 14° C. The ds cDNA is isolated asabove.

The ds cDNA (100 ng in 40 μl) is tailed with dCTP (0.9 mM) in 200 mMpotassium cacodylate, pH 6.9, 1 mM CoCl₂ and 5 mg/ml BSA with 30 unitsof terminal transferase for 60 min at 37° C., followed by heatinactivation. This dC-tailed cDNA is annealed to dG-tailed, PstI cutpBR322 (BRL) in 50 μl TE buffer/0.15 M NaCl at total DNA concentrationsof 0.5 μg/ml DNA for 90 min at 58° C. CaCl₂ treated E. coli MC1061 aretransformed with this vector. The cells are plated and handled at highdensity on nitrocellulose filters laid on agar plates as described by D.Hanahan and M. Meselson [Methods Enzymol. 100, 333-342 (1983)].

An oligodeoxynucleotide mixture is synthesized on the basis of the knownpartial amino acid sequence of Example 3.2, namely the sequenceGlu-Val-Asp-Ile-Ala-Lys-Ala. (SEQ ID NO.13) The 20-meroligodeoxynucleotide mixture of the composition5′-GCYTTIGCQATRTCIACYTC-3′, (SEQ ID NO.14) wherein A, T, G, C and Istand for adenosine, thymidine, guanosine, cytosine and inosine,respectively, Y and R for pyrimidines (T, C) and purines (A, C),respectively, and Q for A, G and T, is synthesized following theprocedure of Y. Ike et al., Nucleic Acid Research 11, 477 (1983). The 5′ends of the oligodeoxynucleotides are rendered radioactive usingγ-³²P-dATP (5000 Ci/mmol) and polynucleotide kinase (Pharmacia) to2-5×10⁸ cpm/μg using standard procedures [T. Maniatis, E. F. Fritsch andJ. Sambrook, “Molecular cloning, a laboratory manual”, Cold SpringHarbor Laboratory, 1982].

Duplicate replicas of the bacterial clones of the cDNA library arehybridized with the above nucleotide mixture following the method ofHanahan and Meselson [loc. cit.] in a medium containing 6×SSC, 5×Denhardt solution, 250 μg/ml tRNA, 50 units/ml heparin and 0.1% SDS at47° C. After hybridization, the filters are washed four times in 6×SSCand 0.5% SDS for 20 min at 20° C. and 5 min at 47° C.

Clone B1,1 containing a DNA plasmid with an insert of approximately 850base pairs is found to hybridize with the oligonucleotide probe and isgrown in LB medium supplemented with 15 μg/ml tetracycline at 37° C.

EXAMPLE 6 Isolation of Plasmid DNA

800 ml of LB medium supplemented with 15 μg/ml tetracycline isinoculated with 1 ml of clone B1,1 (Example 5) and cultured at 37° C. toan optical density OD₅₅₀ of 0.7 (approx. 5 h). 200 μg/ml chloramphenicoldissolved in ethanol are added and culturing continued at 37° C.overnight. The mixture is centrifuged for 20 min at 0° C. with 4000 rpm,the bacterial pellet resuspended in 36 ml TE buffer and transferred toSS34 tubes. The suspension is centrifuged for 5 min at 0° C. with 5000rpm. The pellet is resuspended in 7.5 ml 25% sucrose/50 mM Tris.HCl, pH7.5, treated with 0.75 ml of freshly prepared lysozyme (10 mg/ml in 250mM Tris.HCl, pH 7.5) and incubated for 5 min on ice. 3.0 ml 0.25 M EDTA,pH 8.0, and, after 5 min, 12 ml Triton-Sol (0.1% Triton X-100® [Sigma],60 mM EDTA, 50 mM Tris.HCl, pH 8.0) are added and the incubationcontinued for 1 h at 0° C. The mixture is centrifuged in a SS34centrifuge at 18,000 rpm for 50 min. The supernatant is carefully pouredin a measuring cylinder and the volume adjusted to 30 ml with TE buffer.30 g CsCl and 2.58 ml ethidium bromide (10 mg/ml) are added and themixture centrifuged for 16 h at 20° C. with 48,000 rpm in a VTi 50centrifuge. The lower band consisting of supercoiled DNA is collected,extracted 5 times with isopropanol saturated with aqueous CsCl, anddiluted with TE buffer to remove turbidity. The DNA is precipitated withethanol at −20° C., then purified once more in a CsCl gradient as above.

EXAMPLE 7 Tests Proving that the Selected Clone Codes for the 78 kDaInterferon-induced Protein

7.1 Northern Blot:

Total RNA from IFN-induced human embryonic foreskin cells isolatedaccording to Examples 4.1 and 4.2 and total RNA from corresponding cellsnot induced with interferon are denatured with 1 M glyoxal in 50% (v/v)dimethyl sulfoxide and 10 mM sodium phosphate buffer, pH 7.0,electrophoresed on 1.1% agarose gel and transferred to nitrocelluloseusing 3 M NaCl/0.3 M trisodium citrate essentially as described by P. S.Thomas [Proc. Natl. Acad. Sci. USA 77, 5201-5205 (1980)]. Thenitrocellulose filters are baked for 2 h at 80° C. under vacuum,prehybridized in a buffer containing 5×SSC/50% formamide for 3 h at 42°C., then hybridized for 20 h at 42° C. in the same buffer containingdextran sulfate 500 and 0.5-1.0×10⁶ cpm/ml of DNA from clone B1,1(Example 6) labelled with γ-³²P-dATP and polynucleotide kinase asdescribed above. The filters are washed four times 5 min at 20° C. in2×SSC/0.1% SDS and twice 20 min at 50° C. in 0.1×SSC/0.1% SDS. The dryfilters are exposed to Kodak XAR film with a Cawo intensifying screenfor 6 days at −70° C.

The DNA of clone B1,1 hybridizes to an RNA of approximately 23Scorresponding in size to the expected mRNA coding for the 78 kDAinterferon-induced protein. This mRNA is detected only ininterferon-induced cells.

7.2 Hybrid Selected Translation

10 μg plasmid DNA of clone B1,1 (Example 6) in 20 μl H₂O are heated to100° C. for 10 min, cooled quickly in ice, treated with 20 μl 1 M NaOHand incubated at room temperature for 20 min. The DNA sample isneutralized with 20 μl of a solution of 1M NaCl, 0.3 M trisodiumcitrate, 0.5 M Tris.HCl and 1M HCl, then spotted on a nitrocellulosefilter (3×6 mm, Millipore HAWP). The filter is dried at 20° C. and bakedfor 2 h at 80° C. in a vacuum oven. The filter is placed in asiliconized 1.5 ml Eppendorf tube, treated with 1 ml H₂O, heated in aboiling water bath for 1 min and cooled in ice. The water is removed and50 μl of a solution containing 100 μg total mRNA from IFN-induced cells(Example 4.2) in 0.9 M NaCl, 0.2% SDS, 1 mM EDTA and 20 mM PIPES(1,4-piperazine-diethanesulfonic acid, pH 6.4) added. The filter isincubated for 6 h at 37° C. with constant agitation, then washed fivetimes in 1 ml washing buffer consisting of 50% formamide, 20 mM NaCl, 8mM trisodium citrate, 1 mM EDTA and 0.5% SDS for 15 min at 37° C. Thehybridized mRNA is eluted with 100 μl 1 mM EDTA containing 10 μg tRNA ina boiling water bath for 1 min. The solution is frozen by plunging intodry ice, thawed on ice, and the filter removed. 7 μl 3 M sodium acetateare added and the mixture extracted with phenol/chloroform/isoamylalcohol (1:1:0.04 v/v). 250 μl ethanol are added to the aqueous phase toprecipitate the mRNA.

The eluted mRNA is translated in reticulocyte lysate (AmershamInternational No. N90) according to the instructions of themanufacturer. An aliquot of the proteins synthesized in vitro isseparated by polyacrylamide gel electrophoresis, and radioactiveproteins (from ³⁵S-methionine in the translation system) detected byfluorography. Another aliquot of proteins is immunoprecipitated withmonoclonal antibodies specific for the 78 kDa protein of Example 13. Theimmunoprecipitate is also separated by polyacrylamide gelelectrophoresis and detected by fluorography.

The mRNA selected by the hybridization with DNA from clone B1,1 directsthe synthesis of a protein with the same apparent molecular weight andthe same antigenic properties as the 78 kDa protein isolated fromIFN-induced Namalwa cells (Example 2).

EXAMPLE 8 Subcloning of Plasmid DNA into an M13 Vector

The plasmid DNA of clone B1,1 of Example 6 is fragmented with therestriction enzyme PstI (Boehringer-Mannheim) according to themanufacturer's instructions. The insert is isolated and precipitatedwith ethanol.

Bluescript M13 vector (Stratagene) is cut with PstI. 20 μg vector DNAare dephosphorylated in 50 μl solution containing 8 units calf alcalineintestinal phosphatase, 100 mM glycine, pH 10.5, 1 mM MgCl₂ and 1 mMZnCl₂. The vector DNA is isolated and purified by phenol/chloroformextraction.

0.5 μg cDNA of clone B1,1 and 1.5 μg M13 vector DNA are ligated byincubation for 5 h at 23° C. in 20 μl ligation buffer containing 5 unitsT₄ DNA ligase and 0.5 mM ATP. CaCl₂ treated E. coli rec^(A−) JM109 aretransformed with this DNA solution. The cells are plated on LB platescontaining 100 μg/ml ampicillin, 40 μg/ml X-Gal(5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) and 5 mM IPTG(isopropyl β-D-thiogalactopyranoside). The colonies are grown overnightat 37° C., and transformants selected by white color from blue cellplaques containing unchanged M13 vector.

Selected individual colonies are grown in 1 ml of LB medium containing50 μg/ml ampicillin overnight at 37° C. After centrifugation thesupernatant is discarded and the pellet suspended in 100 μl 50 mMglucose, 25 mM Tris.HCl, pH 8.0, and 10 mM EDTA. After 5 min at 22° C.,200 μl 0.2 N NaOH/1% SDS are added, the mixture incubated at 0° C. for 5min, treated with 150 μl precooled 3 M sodium acetate, pH 4.8, and keptat 0° C. for another 5 min. The mixture is centrifuged in an Eppendorftube for 1 min. 1 ml ethanol is added to the supernatant, and themixture, after 2 min at 20° C., centrifuged again for 1 min. The pelletis washed with 80% ethanol and resuspended in 100 μl 300 mM sodiumacetate. 300 μl ethanol are added, and the mixture kept at −80° C. for30 min, then centrifuged. The pellet is washed with 80% ethanol, driedand suspended in 15 μl TE buffer.

2 μl of this DNA suspension are digested with PstI. Another sample of 2μl is double digested with SacI and HindI. The obtained restrictionfragments are analyzed by electrophoresis on 7% polyacrylamide gels inorder to determine the orientation of the cDNA insert in the vector.

EXAMPLE 9 Subcloning of Plasmid DNA After Unidirectional Deletions

Plasmids from clones of Example 8 containing the cDNA insert in eitherdirection are isolated using the method described in Example 6 exceptthat the clones are cultured in LB medium containing 100 μg/ml ofampicillin instead of tetracycline, and no chloramphenicol is added.

The plasmid DNA is digested to completion with KpnI and HindIII, thenextracted with phenol. 18 μg of this double digested DNA in 300 μl 50 mMTris.HCl, pH 8, 5 mM MgCl₂, 10 μg/ml tRNA, 20 mM 2-mercaptoethanolcontaining 900 units of exonuclease Exo III are incubated at 23° C. 50μl aliquots are removed from the reaction mixture every minute up to 6min, added to a tube with 80 μl 5× concentrated mung bean nucleasebuffer and 270 pi water and frozen in dry ice. The aliquots are heatedat 68° C. for 15 min, then treated with 9 units of mung bean nuclease inmung bean nuclease buffer for 30 min at 30° C. The reaction is quenchedwith 400 μl of buffer-equilibrated phenol/chloroform per aliquot, andthe DNAs isolated by ethanol precipitation.

These DNAs are re-ligated, and the hybrid vectors obtained used totransform E. coli Rec^(A−) JM109 as described in Example 8.Transformants are grown in LB medium containing 100 μg/ml ampicillinovernight at 37° C. Plasmid DNA is isolated and purified in a CsClgradient as described in Example 6.

EXAMPLE 10 Determination of the DNA Sequence

The sequence is determined on the DNAs of Example 6 and Example 9 withthe ²⁰-mer oligonucleotide mixture of Example 5 as a primer followingstandard procedures (dideoxynucleotide method). The partial sequence offormula II is confirmed by upstream and downstream sequencing using asecond primer of the formula 5′-CAGCCACCATTCCAAGG-3′ (SEQ ID NO.15) anda third primer of the formula 5′-CGCACCTTCTCCTCATACTGG-3′ (SEQ ID NO.16)synthesized according to Y. Ike et al. [Nucleic Acid Research 11, 477(1983)].

In brief, 5 μg of plasmid DNA of Example 6 or 9 are linearized with therestriction enzyme PstI (Boehringer-Mannheim) according to themanufacturer's instructions. The DNA is precipitated with 3 volumes ofethanol, then dissolved in 25 μl TE buffer. 8 μl of this solution and 2μl TE buffer containing 0.5 nmol/ml of the primer are mixed, placed in aboiling water bath for 3 min, then frozen by plunging into dry ice. 1 μof 0.1 M Tris.HCl/50 mM MgCl₂, pH 7.4, is added and the mixtureincubated for 30 min at 42° C. This primer/template mixture is treatedwith dNTP mix, α-³⁵S-dATP, Klenow fragment and the dideoxynucleotidesddATP, ddCTP, ddGTP, ddTTP, respectively, following standard procedures[J. R. Dillon, A. Nasim and E. R. Nestmann, “Recombinant DNAmethodology”, Wiley 1985, p. 90-94]. The DNA is denatured and loadedimmediately onto a sequencing 6% polyacrylamide 7 M urea gel [J. R.Dillon et al., loc. cit., p. 89] and the gel run with 90 nM Trisborate/1 mM EDTA, pH 8.3.

The ATG at position 1 of formula II is most certainly the initiationcodon for the protein, since upstream sequences contain terminationcodons at positions −75 (TGA), −65 (TAA), −57 (TGA) and −41 (TGA).

EXAMPLE 11 Preparation of Hybridoma Cells

11.1 Immunization protocol:

5 μg portions of the purified protein (Example 2) are dissolved in 20 μlof 2 M urea solution containing 0.1% SDS and 50 mM mercaptoethanol. Anitrocellulose piece 5×5 mm containing 5 μg protein is implanted intothe peritoneal cavity of a female HR-mouse [obtained from Dr. Biozzi,Institute Curie, Paris, see L. Boumsell & A. Bernard, J. Immunol.Methods 38, 225 (1980)]. Four weeks later 5 μg 78 kDa protein inincomplete Freund's adjuvant containing 50 μg adjuvant peptide (Sigma)are injected intraperitoneally (i.p.), and three bi-weekly boosterimmunizations with the same sample compositions are given i.p. Afterfour weeks, serum is collected and the antibody titer to the 78 kDaprotein determined by the dot-immunoassay of Example 12. Mice with highantibody titer are further immunized by two more bi-weekly injectionsand a final booster immunization one week later. After three days thespleen is taken for the fusion.

11.2 Cell Fusion:

All fusion experiments are performed according to the procedure of G.Köhler and C. Milstein [Nature 256, 495 (1975)] using the nonsecretingSp2/0-Ag14 myeloma line [M. Shulman, C. D. Wilde and G. Köhler, Nature276, 269 (1978)]. 10⁸ spleen cells are mixed with 10⁷ myeloma cells inthe presence of 1 ml of 50% polyethylene glycol (PEG 1500, Serva). Afterwashing, the cells are resuspended in 48 ml of standard Dulbecco'sminimum essential medium (Gibco No. 0422501). 3×10⁶ normal mouseperitoneal exsudate cells per fusion are added as feeder cells. Thecells are distributed into 48×1 ml Costar wells and fed 3 times per weekwith standard HAT selection medium for 3 to 6 weeks. When the growth ofhybridoma cells becomes visible, the supernatants are screened by thedot-immunoassay of Example 12. The hybridoma cells are cloned bylimiting dilution in microtiter plates at least once, then passagedthrough HR mice by i.p. injection. Hybridoma cells are harvested fromascites and cloned once more by limiting dilution. The nine hybridomasselected for further studies are particularly stable and secrete largequantities of immunoglobulin. They are designated 885 S35.8.1, 885S35.16.11, 885 S56.55.7.12.48, 885 S56.55.7.21.25, 885 S56.55.7.27.5,885 S56.55.7.27.11, 885 S56.55.13, 885 S56.55.17, and 885 S56.67.15.

EXAMPLE 12 Dot-immunoassay for Antibody Screening

The purified protein of Example 2 is dissolved in 2 M urea, 0.1% SDS and50 mM mercaptoethanol. The dilutions of the protein are made in TBScontaining 10%. inactivated horse serum. The protein is applied in theform of dots (0.2 μl) onto nitrocellulose (type HAWG from MilliporeCorp., Bedford, Mass.). The dilutions of antibodies from mouse serum orhybridoma culture medium are made in TBS containing 10% inactivatedhorse serum. The dot immunobinding assay, a modified enzyme-linkedimmunosorbent assay, is carried out using a rabbit anti-mouse IgGperoxidase conjugated second antibody and H₂O₂/4-chloro-1-naphthol inTBS as described by M. M. Derer et al. [J. Allergy Clin.Immunol. 74, 85(1984)].

EXAMPLE 13 Isolation and Purification of Monoclonal Antibodies

13.1 In Vivo Synthesis:

Balb/c mice 8-10 weeks of age (Tierfarm Sisseln, Switzerland) arepretreated intraperitoneally with 0.3 ml pristane (Aldrich). 2-3 weekslater, 2-5×10⁶ cloned hybridoma cells and 0.2 ml pristane are inoculatedintraperitoneally. After 8-10 days ascites fluid is collected,centrifuged at 800×g and stored at −20° C.

Defrosted ascites fluid is centrifuged at 50,000×g for 60 min. A fatlayer floating on the surface is carefully removed, and the proteinconcentration is adjusted to a concentration of 10-12 mg/ml. Crudeimmunoglobulin is precipitated by dropwise addition of 0.9 volumeequivalents of saturated ammonium sulphate at 0° C., then dissolved in20 mM Tris.HCl/50 mM NaCl (pH 7.9) and dialyzed against the same buffer.An immunoglobulin fraction is obtained by DEAE-D52 cellulose (Whatman)chromatography using a buffer gradient system of 20 mM Tris.HCl/25-400mM NaCl, pH 7.9. The immunoglobulin is again precipitated with ammoniumsulphate and dissolved in PBS at a concentration of 10 mg/ml.

SDS polyacrylamide gel electrophoresis demonstrates a purity grade ofmore than 95 percent for all the monoclonal antibodies.

13.2 In Vitro Synthesis:

A preculture of a cell line of Example 11.2 is obtained by culturinghybridoma cells at physiological temperature (around 37° C.) in RPMI1640 medium containing 10% FCS to a final cell density of 5×10⁵ to 10⁶cells per ml. The whole preculture is filled into Bellco culture vesselsand adjusted to a total volume of 1500 ml with fresh RPMI 1640medium/10% FCS. The culture is stirred at around 37° C. under 5% CO₂ at30 rpm for two to three days, then diluted to a total volume of 3000 mlwith RPMI 1640/10% FCS and stirred for another seven to ten days. Afterthis time 95% of the cells are dead. The culture broth is centrifuged at1000×g for 20 min at 4° C. The supernatant is filtered through a filterwith pore size 0.2 μm under sterile conditions. Crude immunoglobulin isprecipitated by slow dropwise addition of 0.9 volume equivalents ofsaturated ammonium sulfate at 0° C. This precipitate is purified asdescribed in Example 13.1 and gives monoclonal antibodies with a purityof 95% or more.

EXAMPLE 14 Characterization of Monoclonal Antibodies

14.1 Determination of Class and Subclass of Monoclonal Antibodies:

The class and subclass of monoclonal antibodies produced by clonedhybridoma cells is determined by the known agar-gel immunodiffusiontechnique of Ouchterlony using class and subclass specific rabbitantibodies (Bionetics). The results are confirmed by an enzymeimmunoassay (ELISA) in the following way: Microtiter plates are coatedwith 1 μg per well of a rabbit immunoglobulin preparation of a class- orsubclass-specific serum (Bionetics) in 50 μl of PBS. Free bindingcapacity of the plate is saturated with a buffer of 1% bovine serumalbumin in PBS containing 0.2% NaN₃ (w/v), pH 7.4. 100 μl probescontaining monoclonal antibodies are incubated in the wells at 37° C.for 1 h. The plates are washed with PBS, then incubated at 37° C. for 1h with a phosphatase conjugated rabbit immunoglobulin preparation of thesame specificity as used for coating the plates. The fixed enzyme isdeveloped by incubating (37° C., 30 min) with a solution of the enzymesubstrate p-nitrophenyl phosphate (1 mg/ml in diethanolamine buffer 10%containing 0.5 mM MgCl₂ and 0.02% (w/v) NaN₃, pH 9.8) and measuring theoptical density at 405 nm. The monoclonal antibodies 885 S35.8.1, 885S35.16.11, 885 S56.55.7.12.48, 885 S56.55.7.21.25, 885 S56.55.7.27.5,885 S56.55.7.27.11, 885 S56.55.13, 885 S56.55.17, and 885 S56.67.15. allbelong to the class IgG₁.

14.2 Selectivity Towards Human 78 kDA Protein:

Mouse A2G embryonic diploid cells, rat embryonic diploid cells, hamsterembryonic diploid cells, horse kidney diploid cells and cells of thedermis cell line NBL-6, ATCC No. CCL57, calf kidney diploid cells, catembryonic lung diploid cells, monkey cells of the Vero cell line ATCCNo. CCL81, rabbit embryonic cells, sheep choroid plexus cells, and pigkidney diploid cells and cells of the kidney cell line PK-15, ATCC No.CCL33 are incubated with recombinant interferon α/B-D hybrid asdescribed for human cells in Example 1.2 and 4.1 (M. A. Horisberger & K.de Staritzky, J. gen. Virol. (1987), Vol. 68). In the cells of all ofthese species at least one protein related to the human 78 kDa proteinis detected. These antigenically related proteins are identified with apolyclonal antibody serum obtained from mice immunized with human 78 kDAprotein according to Example 11.1. However, the monoclonal antibodies885 S35.8.1, 885 S56.55.13 and 885 S56.67.15 bind only to human 78 kDaprotein and not to the related proteins of the species mentioned whentested in the immunofluorescence test of Example 17, theimmunoprecipitation test of Example 18, or in a Western blot. For theWestern blot, the proteins are separated by SDS polyacrylamide gelelectrophoresis and transferred to nitrocellulose, then tested asdescribed for the immunodot assay of Example 16.

EXAMPLE 15 Enzyme-immunoassay (ELISA)

15.1 Labelling of Monoclonal Antibody 885 S35.8.1 With AlkalinePhosphatase:

1.4 mg of monoclonal antibody 885 S35.8.1 in 1.4 ml of PBS are coupledfor 2 h with a solution containing 5 mg of alkaline phosphatase (SigmaP6774, type VII-T) according to the standard method of Voller et al.[Bull. World Health Organ. 53, 55 (1976)] using glutaraldehyde (0.2%v/v). The conjugate is transferred into 5 ml of Tris buffer 0.05 M, pH8.0, containing 1 mM MgCl₂, 1% BSA and 0.02% NaN₃. The solution is keptin the dark at 4° C.

15.2 Assay Procedure:

Polypropylene Microtitre Plates (Dynatech Labs. Inc.) are coated over aperiod of 2 h at 37° C. and overnight at 4° C. with 150 μl of a solutionof the monoclonal antibody 885 S56.55.13 (10 μg/ml) in a buffer pH 8.6(carbonate-buffered 0.9% saline containing 0.02% sodium azide). Theplates are washed five times with PBS, and protein-reactive sites stillpresent are saturated by incubation for 1 h at 37° C. with 250 μl of abuffer pH 7.4 (0.2% gelatine and 0.2% NaN₃ in PBS). Plates coated inthis manner can be kept at 4° C. in this buffer for a few days.

50 μl of a dilution series of a test solution or a standard solutioncontaining the 78 kDa protein, 50 μl of buffer pH 7.4 and 50 μl of asolution of the phosphatase-labelled antibody 885 S35.8.1 (Example 15.1)diluted 1:100 with buffer pH 7.4 are mixed and incubated in the wells ofthe microtiter plates for 2 h at 37° C. and for 30 minutes at 4° C. Theplates are washed five times with PBS, then incubated for 30 min at 37°C. with 150 μl of a solution of p-nitrophenyl phosphate (1 mg/ml in 10%diethanolamine buffer, 0.5 mM MgCl₂, pH 9.8). By measuring the opticaldensity at 405 nm, the amount of released p-nitrophenol is determined,which is proportional to the amount of the bound enzyme phosphatase andhence proportional to the amount of the 78 kDa protein in the testsolution.

Similar results are obtained, when the microtiter plates are coated withmonoclonal antibody 885 S35.8.1 or 885 S56.67.15 and phosphatase-coupledmonoclonal antibody 885 S56.55.13 is used as second antibody.

15.3 Test Kit for ELISA:

A test kit for the assay described in Example 15.2 contains

polypropylene microtiter plates,  20 ml of monoclonal antibody 885S56.55.13 (10 μg/ml) in carbo- nate-buffered saline (0.9% NaCl, 0.42%NaHCO₃, 0.0072% Na₂CO₃, 0.02% NaN₃)  1 ml of alkalinephosphatase-coupled monoclonal antibody 885 S35.8.1 (Example 15.1, 0.3mg antibody per ml) in Tris buffer (0.05 M, 1 mM MgCl₂, 1% BSA, 0.02%NaN₃, pH 8.0)  2 ml standard solution containing 5 μg 78 kDa protein 300ml PBS 300 ml buffer pH 7.4 (0.2% gelatine and 0.2% NaN₃ in PBS)  50 mlof p-nitrophenyl phosphate (1 mg/ml) in diethanolamine buffer (10%, 0.5mM MgCl₂, 0.02% NaN₃, adjusted to pH 8.9 with HCl) calibration curvecolour intensity scale instruction manual

EXAMPLE 16 Immunodot Assay

16.1 Assay Procedure:

A dilution series of the solution to be tested for the presence of the78 kDa protein and of a standard solution are prepared in TBS containing10% inactivated horse serum. The dilutions are applied in the form ofdots (0.2 μl) onto nitrocellulose (type HAWG, Millipore Corp., Bredford,Mass.). The excess protein-binding capacity of the nitrocellulose isblocked by incubating the nitrocellulose for 2 h at 37° C. in TBScontaining 10% horse serum. The nitrocellulose is cut into suitablestrips, then incubated with solutions of the monoclonal antibody 885S56.55.13 or 885 S35.8.1 (2 μg/ml and 10 μg/ml) in TBS for 2 h at roomtemperature. The strips are washed five times in TBS and furtherincubated for 2 h in a 10,000-fold dilution of a rabbit anti-mouse IgGperoxidase conjugated second antibody, washed five times in TBS, thendeveloped in a freshly mixed peroxidase substrate solution consisting of0.6 volumes of 4-chloro-1-naphthol (3 mg/ml in methanol), 10 volumes ofTBS and 0.004 volumes of 30% hydrogen peroxide for 15 min at roomtemperature. If desired the spots can be scanned with a reflectancedensitometer at 600 nm (CAMAG, Muttenz, Switzerland).

16.2 Test Kit for Immunodot Assay:

A test kit for the assay described in Example 16.1 contains

Nitrocellulose sheets  20 ml of monoclonal antibody 885 S56.55.13 (10μg/ml) in TBS containing 10% horse serum  1 ml of a 1:100 dilution ofrabbit anti-mouse IgG conjugated to horseradish peroxidase in TBScontaining 10% horse serum  2 ml standard solution containing 5 μg 78kDa protein 300 ml TBS 300 ml TBS containing 10% horse serum  10 ml4-chloro-1-naphthol (3 mg/ml in methanol)  10 ml 30% hydrogen peroxideinstruction manual

EXAMPLE 17 Immunofluorescence Test

Cells to be tested for the presence of the protein of the invention aregrown on plastic coverslips. Alternatively, freshly isolated cells fromhuman blood, e.g. lymphocytes or monocytes, are attached by cytospincentrifugation to glass slides pretreated with poly-D-lysine.

The cells are washed with PBS, fixed at 20° C. for 10 min with 3%aqueous paraformaldehyde, permeabilized for 5 min with 0.5% TritonX-100®, washed once more with PBS, and incubated with a solution of themonoclonal antibody 885 S56.55.13 (10 μg/ml) in PBS for 60 min at 37° C.The cells are washed with PBS, treated with a solution offluorescein-conjugated rabbit anti-mouse IgG (DAKO, diluted 1:40 in PBScontaining 5%, horse serum), washed with PBS, and mounted as describedby Johnson et al. [J. Immunol. Methods 43, 349 (1981)]. UV fluorescencemicroscopy reveals the presence of the protein of the invention by abright fluorescence in the cytoplasm of the cells.

EXAMPLE 18 Immunoprecipitation Test for Cells Induced With Interferon

Cells grown in culture or cells freshly isolated from human blood aremounted on plastic or glass plates as described in Example 17. The cellsare treated with a solution of recombinant interferon 5₁ (α/B type) at aconcentration of 5000 international units per ml for 4 h at 37° C., thenincubated for 30 min at 37° C. with 50 μCi per ml of ³⁵S-methionine inHank's balanced salt solution containing sodium bicarbonate, bufferedwith 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES,pH 7.4). The cells are washed with PBS, scraped off the plates,collected by centrifugation, suspended in hypotonic buffer consisting of5 mM Tris pH 7.4, 1.5 mM KCl and 2.5 mM MgCl2 for 5 min, and collectedagain by centrifugation. The cells are lysed with the hypotonic buffercontaining 1% Triton X-100® and 1% deoxycholate for 5 min at 4° C., thencentrifuged at 12,000 rpm for 5 min. Sodium dodecyl sulfate is added tothe supernatant at a final concentration of 0.5%. 6 μl of this solutionand 20 μl of a buffer consisting of 10 mM Tris.HCl, pH 7.4, and 50 mMNaCl (saturated with phenylmethylsulfonyl fluoride) are mixed andrecentrifuged at 12,000 rpm for 5 min. 20 μl of the supernatant and 1 μlof a solution of the monoclonal antibody 885 S56.55.13 (40 μg/ml) in PBScontaining 0.5%. BSA are incubated for 3 h at 4° C., then mixed with 20μl of a 50% (v/v) suspension of Protein A-Sepha-rose®. The sepharosebeads are washed with 500 μl of a buffer consisting of 10 mM Tris pH7.4, 50 mM NaCl, 1 M sucrose, 0.5% deoxycholate and 0.5% Triton X-100®,and the antigen/antibody complex eluted with 30 μl of sample buffer [U.K. Laemmli & M. Favre, J. Mol. Biol. 80, 575 (1973)]. The eluate isanalyzed by one-dimensional SDS gel electrophoresis on 12%polyacrylamide gels in the usual way. The presence of the protein of theinvention at an apparent molecular weight of 78 kDa is revealed byfluorography.

EXAMPLE 19

Surveillance of Interferon Therapy in Humans

Blood samples are taken from patients receiving 10⁷ international unitsof recombinant human interferon alpha (β₂) subcutaneously 24 h and 48 hafter injection. The lymphocytes are purified by centrifugation onFicoll 400 (Pharmacia) density gradient. 4.5×10⁶ lymphocytes aresuspended in 400 μl H₂O, then precipitated with 800 μl ethanol. Thepellet is dissolved in dissociation buffer and separated byone-dimensional SDS polyacrylamide gel electrophoresis. The proteins aretransferred onto nitrocellulose and the 78 kDa protein detected andquantified as described in Example 16.

Compared to patients before interferon treatment and to healthy humans,the level of the 78 kDa protein is increased fivefold at 24 h and 4.8 hafter s.c. interferon α₂ injection.

EXAMPLE 20 Pharmaceutical Preparation for Parenteral Application

200 μg of the 78 kDa protein are dissolved in 3 ml of 5N human serumalbumin. The resulting solution is passed through a bacteriologicalfilter and the filtered solution subdivided under aseptic conditionsinto 10 vials. The vials are preferably stored in the cold, for exampleat −20° C.

What is claimed is:
 1. A substantially pure protein, wherein saidprotein is present in Namalwa cells treated with interferon α or β, hasa molecular weight of approximately 78 kDa as determined by sodiumdodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), has anisoelectric point of approximately 6.3, and comprises (SEQ ID NO:1. 2.The protein of claim 1, that comprises the sequence of SEQ ID NO:2. 3.The protein of claim 1, wherein said Namalwa cells are treated with arecombinant interferon α/β, αD, α/F or interferon α/B-D hybrids.
 4. Aprotein of claim 1, 2 or 3, characterized by the following range ofamino acid composition: Asx 54-60, Glx 91-101, Ser 37-41, Thr 30-34, Gly41-46, Ala 45-50, Arg 36-40, Pro 24-28, Val 38-42, Met 17-19, Ile 41-46,Leu 64-72, Trp 0-3, Phe 24-27, Cys 5-7, Lys 45-50, His 12-14, and Tyr11-13.